RABBIT MUSCLE PYRUVATE KINASE:
STRUCTURAL AND CATALYTIC STUDIES

Thesis for the Degree of Ph. D‘
MICHIGAN. STATE UNIVERSITY
GEORGE SAMUEL JOHNSON
1969

THE-78:3.

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Date

LIBRARY

Michigan State
Univcrsx'ty

 

 

This is to certify that the
thesis entitled

RABBIT MUSCLE PYRUVATE KINASE:
STRUCTURAL AND CATALYTIC STUDIES

presented by

GEORGE SAMUEL JOHNSON

has been accepted towards fulfillment
of the requirements for

PhD BIOCHEMISTRY

degree in

747(th

Major professor/

AUGUST 25, 1969

 

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ABSTRACT

RABBIT MUSCLE PYRUVATE KINASE:
STRUCTURAL AND CATALYTIC STUDIES

By

George Samuel Johnson

The dissociation of tetrameric rabbit muscle pyruvate kinase
(mol wt = 237,000) into unfolded subunits (mol wt = 57,000) in 6jfl
guanidine has been shown to be reversible. A systematic study of the
factors affecting the reversal of the dissociation led to conditions
where up to 70 9b of the initial catalytic activity was regained.
The reversal procedure consisted of two phases: (1) a 100-fold dilu-
tion of guanidine-dissociated enzyme (0°) into a reversal solvent at
00 and (2) incubation of the resulting solution at a higher tempera-
ture, usually 16o. Conditions for Optimum reversal of dissociation
were (1) pH 8; (2) protein concentration, 0.0h mg/ml; (3) ionic
strength, 0.5; (h) reducing agent, 0.06 M B-mercaptoethanol and
(5) temperature--0o dilution, followed by six hr at 16°. The half-
time for activity recovery was approximately hS min at both 0.0é
mg/ml and 0.09 mg/ml enzyme concentration. Two metabolites, insulin
and phosphate ion, were found to greatly influence the reversal of
dissociation. Insulin decreased the activity recovery upon reversal,
in contrast to what would be expected for an inducer of the enzyme.
Phosphate ion yielded activity recovery at 36°; neglible activity was
recovered at that temperature in its absence. The reversal of disso-

ciation was not affected significantly by the addition of a number of

George Samuel Johnson

metabolites including ATP, ADP, S'-AMP, 3'-5'-AMP, lactate, FDP and
NAD+. The reassociated enzyme had the same Kh, heat stability, and
sedimentation coefficient as the native enzyme.

Rabbit muscle pyruvate kinase was 90 96 inactivated by binding
2-h moles of pyridoxal-5'-phosphate (PLP) per mole of tetrameric enzyme.
Incubation with PLP in 0.2 M_imidazole (pH 7.5) at 250 resulted in a
time-dependent loss of enzymatic activity which reached a final value
in 10-20 min at 250. Half maximal loss of activity occurred with 0.0h
mM_PLP. The inactivation did not cause a gross conformational change.
The inactivation was first order with respect to PLP concentration and
enzyme concentration; the second order rate constant was 37 M71 sec-1
Increasing ionic strength decreased the rate of inactivation by PLP
but low concentrations (1-10 m!) of divalent cations increased it
above the level with no salt. The phosphate containing metabolites,
ADP, ATP, phosphoenolpyruvate and fructose-diphosphate also decreased
the rate; the effect was more pronounced with low concentrations (0.1
mg) of divalent cations. The inactivation was relatively specific for
PLP since various analogues including pyridoxamine, pyridoxamine-S-
phosphate, and pyridoxal caused little or no inactivation. Reduction
with NaBH4 at various concentrations of PLP showed that there were 2
types of binding: (1) a specific, incativating binding, involving 2-h
moles PLP bound per mole enzyme, and (2) a nonspecific, noninactivating
binding which involved at least 20 additional moles PLP bound per mole
enzyme. Both types involved Schiff base formation with é-NHe groups
of lysine. Reversal of the inactivation was accomplished, only with

unreduced enzyme, by dilution, by dialysis, or by addition of Tris.

George Samuel Johnson

The sedimentation coefficient for rabbit muscle pyruvate kinase
in the original native state has been determined to be 8:0,w = 9.6 S -
9.8 S by sucrose density centrifugation of a crude extract from frozen
rabbit muscle. A similar experiment with a crude extract from frozen
rabbit liver yielded a value of 8:0,w = 7.h S and variable amounts of
a faster sedimenting protein (about 9.5 S) for rabbit liver pyruvate
kinase.

The effect of high pH on rabbit muscle pyruvate kinase was
studied with enzyme preparations from fresh rabbit muscle and from
rabbit muscle frozen 2-3 years. The fresh muscle preparation in 0.05
M Tris or glycine buffer, 0.15 M KCl, 0.001 M EDTA showed: (1) a
single peak in the pH region 7.h-10; (2) at either 5° or 2ho, an
initial transition at pH 8.5-9.0 from 826:: 36 = 9.6 to so'is 36 =
9.3 S; (3a) at 5°, a second transition at pH 10.5 and above yielding
a slower sedimenting peak (8.0 S) and an additional peak (h 8); (3b) at
2&0, a second transition at about pH 10.1-10.2, resulting in 2 or more
broad peaks in sedimentation velocity experiments. The enzyme prepared
from the frozen muscle under similar conditions showed different results.
(1) At 2&0, the initial transition occurred at much lower pH (pH 7.9-
8.h). (2) The initial transition yielded a more dissociated or unfolded
enzyme, 826:: ab = 9.h S to 8.7 S, and the peak broadened much more
rapidly during sedimentation velocity experiments. (3) At 2&0, a second
transition (loss of well-defined peaks) occurred at pH 9.8 and above;
0.15 M KCl was required to observe this effect at pH 9.8, but not at

higher pH.

The sedimentation coefficient of rabbit muscle pyruvate kinase

George Samuel Johnson

prepared from fresh muscle was greatly influenced by salt concentration.

It increased from a value of about 9.3 S in no added salt to a maximum

0
value of 8:61: /° = 9.6 s in 0.1 1~_t KCl (pH 7.5, 5° or 2+0) and 0-05 M -
)

0.6 l_4 (NH4)9SO4 (pH 8.0, 50). It then decreased with increasing salt

0
.16
concentration to a value of 520 w b = 7.65 S in 3.0 M KCl. Similar
1

results were obtained with increasing KCl concentrations at pH 10.0 at

21?.

Sedimentation equilibrium analysis showed that the decrease in

sedimentation coefficient was due to a dissociation into dimers or

monomers. The results were also consistent with a dissociation-assoc-

iation rapid equilibrium between monomers (or dimers) and tetramers

and/or octamers .

RABBIT MUSCLE PYRUVATE KINASE:

STRUCTURAL AND CATALYTIC STUDIES

By

George Samuel Johnson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1969

TO MY PARENTS

ii

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ACKNOWLEDGMENTS

The author wishes to express his appreciation to Dr. W. C. Deal,
Jr. for his guidance and encouragement throughout the course of this
study. The author also thanks Dr. L. L. Beiber, Dr. J. E. Varner, and

Dr. C. H. Suelter for reviewing sections of this manuscript prior to

'publication in biochemical journals.

The work of Mr. Y. Y. Shum in aiding with the ultracentrifugal
studies, and the help of Mrs. Karen Hollen, Miss Shirley Ann Williams,
sand my wife, Leslye, in the preparation of this manuscript are greatly
zippreciated. The predoctoral fellowship support of the National Insti-

trutes of Health is gratefully acknowledged (l-Fl-GM-35,507).

iii

VITA

George Samuel Johnson was born in Cokato, Minnesota, on August

25, l9h3, and lived for the first eighteen years of his life on a

small dairy farm near Annandale, Minnesota. He attended high school

at Annandale where he was involved in numerous extracurricular activ-

ities including sports, editor of the school yearbook, and Senior

(Zlass president. At the end of his high school career, he was

elected to the National Honor Society.
Following graduation, George attended Augsburg College in

Minneapolis, Minnesota. His early interests were in engineering, but
under the guidance of Dr. Courtland Agre, he became interested in and

eventually majored in Chemistry, graduating Cum Laude.
George's scientific experience extended through the summer

months. The summer of 1963 was spent working at the USDA Rust Labor-
atory at the University of Minnesota where much work was done in the
The following

growth and identification of wheat and oat rusts.

sumer George deve10ped an interest in Biochemistry and experienced
h is first contact with biochemical research while participating in the

NSF undergraduate research program at the Department of Biochemistry,

University of Minnesota. The research during this program under the
snidance of Dr. Robert Jenness centered on developing procedures com-

bining disc electrOphoresis and inmunoelectrOphoresis.

With a career of medical research in mind, George entered grad-

iv

uate school in the Department of Biochemistry, Michigan State University.
He was awarded an NIH Predoctoral Research Fellowship to complete his
graduate studies. His thesis research, under the guidance of Dr. W. C.
Deal, Jr., was aimed at further understanding the structure and control
of the glycolytic enzyme, pyruvate kinase. George also was accepted
for membership in the American Chemical Society.
George has been awarded an NIH postdoctoral research fellowship
‘to study phospholipid interactions in oncogenic viral infectivity.
flfhis research is to be conducted at the National Institutes of Health
jLn Bethesda, Maryland, under the direction of Dr. Ira Pastan.
He also has been offered a staff position at the University of
Ghana in Accra, Ghana, Africa, and is considering acceptance of this

Iacasition upon completion of the research at NIH.

ORGANIZATION OF THE THESIS

The three areas of research are covered individually in the

three chapters of this thesis. For convenience to the reader each

chapter is presented as an independent entity in the format of a
scientific paper, with its own Abstract, Materials and Methods,
The only deviation

Introduction, Results, and Discussion sections.

from this format is that the references for all three chapters are

(zombined at the end of the thesis.
Chapter One has already been published under the title

"Metabolic Control and Structure of Glycolytic Enzymes VIII.

Reversal of the Dissociation of Rabbit Muscle Pyruvate Kinase into

Unfolded Subunits", by George S. Johnson, Marlene Steinmetz Kayne,

 

and William C.-Deal, Jr. (1969), Biochemistry g, 2155.

A preliminary report of the results of Chapter Two has been

published, Federation Proceedings &, 86+, 1969. The detailed results

have been accepted for publication in the Journal of Biological

Chemistry, under the title, "Specific Inactivation of Rabbit Muscle

Pyrtlvate Kinase by a Specific Binding of 24+ Moles of Pyridoxal 5'-

Ph 0 s phate" .

Chapter Three is also being prepared for publication.

vi

INTRODUCTION .

I.

II.

:[III.

TABLE OF CONTENTS

Approach to the Research . . . . . . .

Literature Review: Pyruvate Kinase . . . . . . . .

A. Introduction . . . . . . . . . . . . . . . . .
B. Reactions Catalyzed by Pyruvate Kinase . . . .
C. The Pyruvate Kinase Reaction . . . . . . .

1. Reaction Equilibrium and Energetics . .

2.

Reaction Mechanism . . . . . . . . . .

D. Pyruvate Kinase Molecular Structure . . . . . .

E. Importance in Glycolysis and Gluconeogenesis

F. Mechanism for Control of Pyruvate Kinase . . .

l.

2.

Control of Muscle Pyruvate Kinase Activity

Control of Pyruvate Kinases from Sources

Other Than Muscle . . .
a. lg_Vivo Studies . . . . . . . . .

b. .Ig'Vitro Studies . . . . . . . . . . .

Literature Review: Pyridoxal Catalysis . . .

A. Discovery of Pyridoxal Catalysis . . . . .

B. Reactions Catalyzed by Pyridoxal Compounds . .

C. General Mechanism for Pyridoxal Catalysis . . .

D. Nonenzymatic Pyridoxal Catalysis . . . . . . .

E. Enzymatic Pyridoxal Catalysis . . . . . . . . .

vii

Page

r-P'KNWKN

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10
10
11
17
17
18
19
21

21

 

TABLE OF CONTENTS--Continued

CHAPTER ONE-~REVERSAL OF THE DISSOCIATION OF RABBIT MUSCLE

II.

III.

IV.

PYRUVATE KINASE INTO UNFOLDED SUBUNITS . . . . . .

Abstract . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . .

A. Effect of pH and Buffer Species . . . . . . . . .
B. Effect of Salt Species and Ionic Strength . . . .
C. Effect of Temperature and Time of Incubation . . .
D. The Phosphate Effect . . . . . . . . . . . . . .
E. Effect of Protein Concentration . . . . . . .

F. Effect of Sucrose . . . . . . . . . . . . . .

G. Half-Times for Reversal . . . . . . . . . . . . .
H. Effect of Reducing Agent Species and Concentration
I. Effect of Added Metabolites . . . . . . . . . . .
J. Comparison of Native and Reversed Enzyme . . . .
Discussion . . . . . . . . . . . . . . . . . . . . .
A. Possible Effects of Metabolites . . . . . . . .
Materials and Methods . . . . . . . . . . . . . .

A. Enzyme Preparation and Assay . . . . . . . . . . .
B. Reagents . . . . . . . . . . . . . . . . . .

C. Dissociation Procedure . . . . . . . . . . . . . .
D. Reversal Procedure . . . . . . . . . . . . . . . .

E. Comparison Studies . . . . . . . . . . . . . . .

viii

Page

25
2’4-
25
27
28
28
28
35
38
39

59
112

1+9
5 1
5 1
61
611
611
65
66
66
67

TABLE OF CONTENTS--Continued
Page
CHAPTER TWO--INACTIVATION OF TETRAMERIC RABBIT MUSCLE PYRUVATE
KINASE BY SPECIFIC BINDING 0F 2-h MOLES OF
PYRIDOXAL 5'-PHOSPHATE . . . . . . . . . . . . . . . 68
I. Abstract . . . . . . . . . . . . . . . . . . . . . . . . 69
II. Introduction . . . . . . . . . . . . . . . . . . . . . . 70
III. Results . . . . . . . . . . . . . . . . . . . . . . . . 71
A. Spectral Analysis . . . . . . . . . . . . . . . . . 7h
B. Reduction with Sodium Borohydride . . . . . . . . . 81
C. Rate of Inactivation . . . . . . . . . . . . . . . . 85
D. Structural Analysis . . . . . . . . . . . . . . . . 99
E. Reversal of the Inactivation . . . .'. . . . . . . . 99
IV. Discussion . . . . . . . . . . . . . . . . . . . . . . . 99
A. Possible Biological Significance . . . . . . . . . . 99
B. Number of Binding Sites . . . . . . . . . . . . . . 102
C. Mechanism of the Inactivation . . . . . . . . . . . 103
V. Methods and Materials. . . . . . . . . . . . . . . . . . 106
A. Inactivation . . . . . . . . . . . . . . . . . . . . 106
B. Reduction . . . . . . . . . . . . . . . . . . . . . 106
C. Centrifugation . . . . . . . . . . . . . . . . . . . 107
D. UV Spectra . . . . . . . . . . . . . . . . . . . . . 107

CHUAI?TER THREE--STRUCTURAL CHANGES OF RABBIT MUSCLE PYRUVATE
KINAS E O O C O O O O O O O O O O O I O O C O C O O 1 O 8

I 0 Abstract 0 O O O I O O O O O O O O O O O O O O O O O O O 109
II. Introduction . . . . . . . . . . . . . . . . . . . . . . 110

IE II. Results . . . . . . . . . . . . . . . . . . . . . . . . 115

ix

 

 

TABLE OF CONTENTS--Continued

A. Analysis of the Sedimentation Properties of
Pyruvate Kinase from Crude Extracts of Rabbit
Skeletal Muscle and Rabbit Liver . . . . . . .

B. Effect of pH on the Sedimentation Coefficient
of Rabbit Muscle Pyruvate Kinase . . . . . . .

C. Effect of Salt Concentration on the Sedimenta-
tion Coefficient of Pyruvate Kinase . .

D. Analysis of Rabbit Muscle Pyruvate Kinase in
5.0 n KClo O O O O O O O O O O O O O O C O

E. Correlation of the Change in Sedimentation
Coefficient with K01 Concentration to Enzy-
matic Activity . . . . . . . . . . . . . . . .

F. Reversal of the Transition . . . . . . . . . . . .

IV. Discussion . . . . . . . . . . . . . . . . . . . . . .

A. Mechanism of Alteration of Structure by High pH

B.

and High Salt 0 o o o o o o o o o o o o o o o e o

The Partial Specific Volume Factor . . . . .

V. Materials and Methods . . . . . . . . . . . . . . . .

A.

H.

Analysis of the Crude Extracts from Rabbit
Muscle and Rabbit Liver . . . . . . . . . . . . .

Preparation of the Enzyme for Ultracentrifugal
AnaIYSis O O O O O O I O O O O O O O O O O O O I O

Ultracentrifugal Analysis . . . . . . . . . . . .
Sedimentation Velocity Analysis . . . . . . . . .
Sedimentation Equilibrium Analysis . . . . . . . .
Diffusion Coefficient Analysis . . . . . . . .

Molecular Weight Determination Using the Svedberg
Equat ion 0 O O O O O O O 0 O O O O I O O O O O O O

Buffer Correction Factors . . . . . . . . . . . .

SIMRYANDCONCLUSIONS....................

Page

115

120

128

1511

1110
1115
1115

11111
1115
1116

1117

1117
1118
1118
1118
1119

1119
150

151

TABLE OF CONTENTS--Continued

Page
APPENDIX I--DERIVATION OF RATE EQUATION USED TO STUDY THE
RATE OF INACTIVATION OF PYRUVATE KINASE BY
PYRIDOXAL 5'-PHOSPHATE . . . . . . . . . . . . . . . 15h
APPENDIX II--DETERMINATION OF THE CORRECTION FACTORS NECES-
SARY FOR CORRECTING THE OBSERVED SEDIMENTATION
COEFFICIENTS To STANDARD CONDITIONS . . . . . . . . 156
I. The Correction Factor . . . . . . . . . . . . . . . . . 156
II. Necessity for Additional Determinations . . . . . . . . 158
III. Results 0 O O 0 O O O O O O O O O O O O O O O O O O O O 158
LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . . . . . 166

xi

 

Table

LIST OF TABLES

Page
Properties of Pyruvate Kinases from Muscle, Liver and
Yeast O O O O O O O I O O O I O O O O O O I O O O O O O 11"
Properties of Pyruvate Kinases from E. Coli, Adipose
Tissue, Erythrocyte and Leukocyte . . . . . . . . . . . 16

Effect of Metabolites on the Reversal in 0.2 M_KC1 . . . . 50
Effect of Insulin on the Reversal. . . . . . . . . . . . . 52

Sodium Borohydride Reduction of Complexes of Pyruvate
Kinase and Pyridoxal Compounds . . . . . . . . . . . . 8h

Effect of Metabolites on the Rate Constants for
Inactivation of Pyruvate Kinase by PLP . . . . . . . . 95

xii

LIST OF FIGURES

Figure Page
1. Effect of pH on the Reversal of Pyruvate Kinase . . . . 30
2. Effect of Ionic Strength on the Reversal of Pyruvate

Kinase . . . . . . . . . . . . . . . . . . . . . . . 32
3. Effect of Temperature and Length of Incubation of

the Reversal of Pyruvate Kinase . . . . . . . . . . 3h
A. Effect of Phosphate Buffer on the Reversal at 360 . . . 37
5. Effect of Enzyme Concentration on the Reversal of

Pyruvate Kinase . . . . . . . . . . . . . . . . . . hl

6. Effect of Enzyme Concentration on the Half-Time of

Reversal of Pyruvate Kinase . . . . . . . . . . . . Ah
7. Half-Times for Reversal at 360 . . . . . . . . . . . . h6'
8. Effect of B-Mercaptoethanol on the Reversal of
Pyruvate Kinase . . . . . . . . . . . . . . . . . . A8
9. Sucrose Density Gradient Centrifugation Pattern of

Native and Reversed Pyruvate Kinase . . . . . . . . 5h

10. Lineweaver-Burk Analysis of Native and Reversed
Enzyme With Respect to ADP . . . . . . . . . . . . . 56

11. Lineweaver-Burk Analysis of Native and Reversed
Enzyme With Respect ot KCl . . . . . . . . . . . . . 58
122. Heat Stability of Native and Reversed Enzymes at 500. . 60

155.. Effect of Pyridoxal-5'-Phosphate (PLP) Concentration
on the Rate of Inactivation of Rabbit Muscle

Pyruvat e Kinas e O O O O O O O O O O O O O O O I O O O 73

1“LL - Effect of PLP Concentration on the Pyruvate Kinase
Activity Obtained at Equilibrium . . . . . . . . . . 76
JLfiE; .. Effect of pH on the Inactivation . . . . . . . . . . . 78

xiii

LIST OF FIGURES--Continued

Figure

16.

17.

l8.

19.

20.

21.

22.

23.

2h.

25.

26.

27.

228.

30.

Absorption Spectra of the Reduced and Nonreduced
Pyruvate Kinase-PLP Complexes . . . . . . . . .

Activity of Pyruvate Kinase as a Function of Moles
of PLP Covalently Reduced Onto It . . . . . . . .

Logarithmic Plots of Pyruvate Kinase Activity With
Time at Varying PLP Concentrations . . . . . .

Determination of the Second-Order Rate Constant From

the Graph of Pseudo First-Order Rate Constants vs.

PLP Concentration . . . . . . . . . . . . . . . .

Effect of Monovalent Cations on the Pseudo First-
Order Rate Constants for Inactivation . . . . . .

Effect of Divalent Cations on the Pseudo First-Order
Rate Constants for Inactivation . . . . . . . . .

Effect of Salts and Substrates on the Rate of
Inactivation I O O O O O O O O O O O C O O O O

Sedimentation Characteristics of Native and Inacti-
vated Pyruvate Kinase . . . . . . . . . . . . . .

Sedimentation Coefficients vs. Molecular Weights for
Perfect Spheres and Globular Proteins . . . . . .

Sedimentation Coefficient of Pyruvate Kinase in a
Crude Extract From Aged, Frozen Rabbit Muscle .

Sedimentation Coefficient of Pyruvate Kinase in a
Crude Extract From Aged, Frozen Rabbit Liver . .

Effect of pH on the Sedimentation Coefficient of
Pyruvate Kinase Isolated From Fresh Rabbit
Muscle . . . . . . . . . . . . . . . . . . . .

Effect of pH on the Sedimentation Coefficient of
Pyruvate Kinase Isolated From Aged, Frozen
Rabbit Musc1e O O O O O O O O O O O O O O O I O O

Sedimentation Velocity Patterns of Pyruvate Kinase
Isolated From Fresh Rabbit Muscle . . . . . . . .

Sedimentation Velocity Patterns of Pyruvate Kinase
Isolated From Aged Frozen Rabbit Muscle . . . .

xiv

Page

80

83

87

89

92

9h

98

101

113

117

119

123

125

127

130

LIST OF FIGURES--Continued

Figure

31.

32.

53-

52+.

55-
36.

57-

Page

Effect of Salt Concentration on the Sedimentation
Coefficient of Pyruvate Kinase Isolated From
Fresh Rabbit Muscle . . . . . . . . . . . . . . . . . 133

Effect of Protein Concentration on the Sedimentation
Coefficient of Pyruvate Kinase Isolated From
Fresh Rabbit Muscle in 3.0 M KCl Using Sucrose
Density Centrifugation . . . . . . . . . . . . . . . 136

Sedimentation Equilibrium Analysis of Rabbit Muscle
Pyruvate Kinase in 3.0 M KCl . . . . . . . . . . . . 139

Effect of Potassium Chloride of the Enzymatic Activ—

ity of Pyruvate Kinase Isolated From Fresh

Rabbit Musc1e O O O O O O O O O O I O O O O O O O O 1M2
Relative Densities of the Salt Solutions . . . . . . . 160

Relative Flow Time of the Salt Solutions Through the
Viscometer . . . . . . . . . . . . . . . . . . . . . 162

Relative Viscosities of Salt Solutions . . . . . . . . 16h

 

LIST OF ABBREVIATIONS

PEP

P.
1

PP.

1
Tricine
Bicine
FDP
PLP

EDTA

phosphoenolpyruvate

orthOphosphate

perphosphate
N-tris(hydroxymethyl)methylglycine
N,N-bis(2-hydroxyethy1)-glycine
fructose-1,6-diphosphate
pyridoxal-5'-phosphate

(ethylenedinitrilo)tetraacetic acid

xvi

INTRODUCTION
APPROACH TO THE RESEARCH

The aim of the research was to better understand the structural

and control prOperties of the important glycolytic enzyme, pyruvate

kinase, isolated from rabbit muscle.

A reasonable point for control of protein biosynthesis is at

the level of folding of the nascent polypeptide chain. By completely

unfolding a purified enzyme i_r_1_ vitro and determining the variables

required for reconstitution of its original molecular structure and

correlating these with in vivo conditions, it was expected that pos-
sible mechanisms for its control might be revealed. The metabolite

NAD"’had been found to be required for the refolding of yeast glycer-

aldehyde-3-phosphate dehydrogenase and it had been postulated that NAD+
might control the synthesis of the enzyme (Deal, 1969).' It seemed

like 1y that such a control mechanism might exist for pyruvate kinase;

it is a key control point in glycolysis. Chapter One thus describes

the elucidation of the variables affecting the refolding and reassembly

of Pyruvate kinase from completely unfolded subunits.

During the study on the effect of various metabolites on the

reVereal process, it was discovered that the important biological co-
factor, pyridoxal 5'-phosphate (PLP) inactivated the enzyme at low con-

centrations (10-5 M). This inactivation was interesting from a

2
a physiological point of view. PLP has long been known to be an impor-

tant cofactor in enzymatic catalysis; it has an _i_g vivo concentration

of about 0.1165 x 10"5 M in rat muscle (Long, 1961). PLP, with its

active aldehyde group, is an excellent reagent for chemical modifica-

tion of proteins through formation of a Schiff base with protein side

chain amino groups. Since little was known about the functional groups

involved in the catalytic mechanism, and the possibility of participa-
tion of an amino group in the catalytic reaction had been postulated

(Mildvan and Cohn, 1966), it seemed likely that an analysis of this

inactivation would give further understanding of the catalytic mechan-

ism of pyruvate kinase. The results of this study are presented in

Chapter Two.
Several different sedimentation coefficients, ranging from

8:0", . 10.17 S to 8.53 8, have been reported for native rabbit muscle

pyruvate kinase (Warner, 1958; Kayne, 1966; Wilson £51., 1967). A

time-dependent transition from. a 10.07 S species to an 8.53 S Species
has been reported (Kayne, 1966) to occur during storage of the purified
enzyme. The mol wt of the subunits of rabbit muscle pyruvate kinase
has been determined to be 57, 000 (Steinmetz and Deal, {1966). The 10 S

8Pecies (mol wt 237,000; Warner, 1958) can thus be assumed to be a

teliramer. However, there is no readily apparent explanation for the

8-5 S species. Additional studies were needed to determine the orig-
inal 351 111°. native state of the enzyme, and determine what signifi-
cance’ if any, could be attributed to the altered, non-native states.
with this in mind we undertook a detailed analysis to~determine the

original native state of the enzyme and possible conditions which

c.
ould alter it. These results are presented in Chapter Three.

 

LITERATURE REVIEW: PYRUVATE KINASE

INTRODUCTION. Pyruvate kinase is a key enzyme in glycolysis and gluco-

 

genesis. It was first discovered by Lohmann and Meyerhof (19311), and
since has been purified and extensively studied. Crystalline prepara-
tions of pyruvate kinase were first obtained from rabbit muscle
(Biicher t 1., 1955; Teitz and Ochoa, 1958), and later from liver

(Tanaka t 1., 1967a). Also highly pure preparations have been

t 1],, 1968; Hunsley and Suelter, 1967).

obtained from yeast (Haeckel
Its properties have been discussed in great detail in an excellent
review by Boyer (1962) and more recently by Kayne (1966) and Hollen-
bers (1969)-

This review emphasizes the more recent studies on the structural
and functional prOperties of pyruvate kinase from rabbit muscle, and

also discusses studies of pyruvate kinases from sources other than

muscle.

REACTIONS CATALYZED BY PYRUVATE KINASE. In the cell, pyruvate kinase
Participates in glucose metabolism by catalyzing the conversion of
phosphoenolpyruvate to pyruvate with the transfer of a ”high energy"

Phosphate to ADP (Reaction (1)).

K+

+
(1) PEP + ADP + H —_—'95 pyruvate + ATP

Mg2+

 

 

In addition, pyruvate kinase has been shown to catalyze two
additional reactions: the phOSphorylation of fluoride ion (Reaction
(2)) (Flavin g£_§l,, 1956; Teitz and Ochoa, 1958) and of hydroxyl-
amine (Reaction (5)) (Kupiecki and Coon, 1960; Cottam gt al., 1968).

H00;
(2) ATP + fluoride ——5 ADP + fluorOphosphate

(5) ATP + hydroxylamine 32339' ATP + phOSphoryl hydroxylamine
lieactions (1), (2), and (5) require a monovalent cation, preferably
f>otassium, for catalysis. Reactions (1) and (2) require Mg2+ or Mne+
laut not Zn2+, whereas Reaction (5) is most rapid with Zn2+ or C02+ but
is not active with Mg2+. Reactions (2) and (5) are essentially irre-
xrersible, and require bicarbonate for catalysis (Boyer g£_§1,, l9h2;
Thietz and Ochoa, 1958; Kupiecki and Coon, 1960).

Pyruvate kinase (reaction (1)) has a specific activity (”moles

substrate cleaved per minute per mg protein) at 250 of about 180
(Steinmetz and Deal, 1966). This corresponds to a turnover number of
10;:3 per second. The reaction is slightly reversible (this will be
discussed in a later section), and the maximum initial velocity of the
fOrWard reaction is 150 to 200 times that of the back reaction (McQuate
ar1c1 IItter, 1959). Pyruvate kinase has a specific activity of 58 or 58,
“fl1‘311» catalyzing reaction (2) or (5), respectively (Kupiecki and Coon,

IS9€SC>) ,

533§Q§L_3:XRUVATE KINASE REACTION. Reaction Equilibrium and Energetics.
The free energy of hydrolysis of the substrate PEP is unusually high;

<3
‘3‘ 15' =-12,8OO cal/mole. This is sufficiently high to phosphorylate

ADP and still allow a large free energy change for the overall reaction

(Mahler and Cordes, 1966).

Because of this large free energy change, the pyruvate kinase

reaction was initially thought to be essentially irreversible (Meyerhof

_Q _L., 1958). The reaction was first shown to be reversible by Lardy
and Ziegler (1911.5). The equilibrium constant for the reaction was

first reported to be K = 2 x 103 at 500, and K = 1.65 x 103 at 20°,
both at an unspecified pH (Meyerhof and Oesper, 19119). This corres-

ponds to a free energy change of about ~1L500 cal/mole for the reac-

tion. McQuate and Utter (1959) later found that it was pH dependent,
decreasing with increasing pH. The equilibrium constants they deter-

mined at 500 were: K = 6.61 x 103 at pH 7.1+, and K = 0.57 x 10:3 at

pH 9.0. in vivo the reaction in various tissues and organisms does
not appear to be at equilibrium under either aerobic or anaerobic con-

ditions (Lowry and Passonneau, 1961L; Williamson, 1965; Hess g1; _al.,

1965).

Reaction Mechanism. The pyruvate kinase reaction appears to involve

a direct transfer of the phOSphoryl group from PEP to ADP; exchange

 

BtI-Id ies with O18 of water show that the reaction occurs without

exchange of the phosphate oxygens with those of substrate or water

(Harrison £32. 91., 1955), and no evidence could be detected for a

phOsphoryl-enzy'me intermediate (Hess _e_t §_l_., 1961). The substrates

PEP and ADP bind independently, and ATP is a competitive inhibitor to
both substrates, presumably by binding at the ADP site and the
Y~Phosphoryl group of the ATP overlapping the PEP binding site

(Remard gt; fl” 1961). Rose (1960) studied the enolization of

 

6

pyruvate in the reverse reaction. His results show that the enolization

(detritiation of a-methyl hydrogens) is 1.8 times as fast as the phos-

+
phorylation, but that the substrate, ATP, and the activators, K and

Mge‘f are required. This suggests that pyruvate, not enolpyruvate,

)
is the true substrate, and that enolization is not the rate limiting

step. Potassium appears to function by altering the protein conforma-
tion (Kayne and Suelter, 1965; Melchior, 1965; Sorger g3; 31., 1965).

The divalent metal activator binds to the enzyme by combining with
imidazole and another ligand which may be an a-amino group or an
atypical sulfhydryl group, and the ternary complex of enzyme-metal-
substrate is the true reactive species (Mildvan and Cohn, 1965; 1966).

From the above results the following mechanism for the reaction

can be formulated (Mildvan and Cohn, 1966): l) The divalent metal ion

binds to the protein acting as a bridge to aid the binding of the two
substrates; 2) the K+ alters the enzyme conformation to aid the bind-

ing or the proper positioning of the substrates for a direct transfer

of the phosphoryl group from PEP to ADP.

PYR UVATE KINAS E MOLECULAR STRUCI‘ URE .

(11101 wt 257,000, (Warner, 1958)), was first reported to dissociate

Rabbit muscle pyruvate kinase

int1'2) subunits of mol wt 150, 000 in 6 M urea (Morawiecki, 1960).
However, more extensive sedimentation studies established that it is

converted into a dimeric species (mol wt 115,000) in low urea concen-

trat ions (LL-8 I_4_) (Steinmetz and Deal, 1966). The molecular species

in 1.5-2.0 _lji_ urea retains most of its catalytic activity, but all

detectable activity is lost in 1|. g urea (Steinmetz and Deal, 1966;

7

Cottam 91 51., 1969). By several types of analysis, the subunits

(mol wt 57,000) appear to be highly similar if not identical in amino

acid composition (Cottam _e_t _a_l_., 1969). There is disagreement as to

the exact "native" molecular state of rabbit muscle pyruvate kinase.

Sedimentation coefficients ranging from 8.5 S to 10.17 S have been

reported (Warner, 1958; Kimberg and Yielding, 1962; Kayne and

Suelter, 1965; 1968; Kayne, 1966; Wilson __t__1., 1967; Cottam _E_a1.,

1969). A transition in sedimentation coefficient from 10.0 S to 8.5 S

during storage of purified enzyme has been observed (Kayne, 1966;

Cottam g_t_ a” 1969).

There is evidence that the molecular weights of pyruvate
kinases from other sources may differ greatly from that of the muscle

enzyme. The molecular weights of pyruvate kinase from rat liver

(Type "L") and yeast have been determined to be 208,000 and 167, 000,

respectively (Tanaka _e_t_ §_1_., 1967a; Kuczenski, R. T., and Suelter,

C. 11., submitted for publication). (See also Table 2.)

IMPORTANCE IN GLYCOLYSIS AND GLUCOGENESIS. It is advantageous for

Pyruvate kinase activity to be regulated to meet the metabolic needs

0f the cell. When the supply of available carbohydrate is increased,

the need for glycolysis is increased; hence pyruvate kinase activity

should increase. However, under conditions of stress or low carbo-

hydrate diet, the necessary glycogen store may be depleted and a tis-

8‘1e or organism may be required to reform necessary glucose via gluco-

genes is. (The term "glucogenesis" is used to designate formation of

gluC—Ose from all precursors other than glycogen (White a 91., 1968).

Since glucogenesis normally involves pyruvate and PEP as

 

8

intermediates, the pyruvate kinase activity must be inhibited under these
conditions. Moreover, because of the thermodynamic barrier hindering PEP
synthesis from pyruvate, additional reactions involving utilization of
'high energy compounds are required to circumvent the pyruvate kinase
reaction.

This unfavorable energy barrier has been overcome by two basic
Inechanisms: use of the energy of ATP and GTP, or the energy of pyro-
phOSphate .

In glucogenic tissues and organisms (those capable of conducting
iglucogenesis) such as liver, kidney, and yeast, pyruvate and four-carbon
(compounds are converted into PEP by a combination of the reactions
(:atalyzed by pyruvate carboxylase (EC 6.h.1.l) (Reaction (h)) and PEP

«:arboxykinase (EC h.l.l.52) (Reaction (5)) (Utter gt 11., 196h).

(h) ATP + pyruvate + C02 + H20 j--9’+ Pi + oxaloactate

(5) GTP + oxaloacetate ——> GDP + PEP + (:02

The enzyme, phosphoenolpyruvate synthetase (Reaction (6)) has
been discovered in Escherichia coli (Cooper and Kornberg, 1965); and
the enzyme, pyruvate-phosphate dikinase (ATP: pyruvate, phosphate
c1iphosphotransferase) (Reaction (7)), has been discovered in bacteria
(Reeves 2; g” 1968; Evans and Wood, 1968), leaves of trOpical grasses

(Iiz112csh and Stack, 1968), and in Entamoeba histo1ytic (Reeves, 1968).

 

 

(6) pyruvate + ATP : , PEP + AMP + P1
(7) AMP + PPi + PEP E 7 ATP + F1 + pyruvate

It is clear that if pyruvate kinase is active in conjunction

9
with either of these reversal systems, the result would be reformation
of pyruvate from the reformed PEP, with a net loss of high energy com-

pounds.

MECHANISM.FOR CONTROL OF PYRUVATE KINASE. Control of Muscle Pyruvate
Kinase Activity. Although mammalian muscle tissue has not been shown
to undergo glucogenesis and glycolytic flux in muscle appears to be
controlled at the level of phosphofructokinase activity (Williamson,
1965), a few inhibitors have been discovered for muscle pyruvate
kinase (see Table 1). It is inhibited by the products of the reaction,
ATP and pyruvate (Reynard gt 31., 1961), by Ca2+ (Boyer, 1962), AMP
(Kerson gg‘g1., 1967), diethylstilbestrol (Kimberg and Yielding, 1962),
and acetyl CoA (Weber g1 g1,, 1967). Computer simulation studies have
been done on mammalian pyruvate kinase in an attempt to organize these
effects and elucidate the control exerted by pyruvate kinase in the
glycolytic pathway (Kerson g; 31., 1967).

A controversy has recently deve10ped concerning the ATP inhibi-
tion of pyruvate kinase. Does ATP directly inhibit the reaction or is
its effect through a chelation of the Mg2+ required for catalysis?

The inhibition of pyruvate kinase at ATP was first discovered
by Meyerhof and Oesper (l9h9). It was later observed by McQuate and
Utter (1959), who attributed the inhibition to a chelation of the
Mg2+ ion. The inhibition was extensively studied by Reynard _£._1.
(1961) who concluded, using kinetic studies, that the ATP inhibition
was competitive with the substrates ADP and PEP with a Ki of 1.2 x

10"4 M, They concluded that the ATP bound at the ADP binding site

with the transferable YLphosphoryl group of the ATP overlapping the

10

PEP site. Mildvan and Cohn (1966) arrived at similar conclusions using
NMR techniques.

Thus it seemed established that ATP was inhibiting by a direct
binding; however, Wood (1968) contended that there was no inhibition
by ATP of muscle pyruvate kinase at high Mge+ concentrations, and that
the inhibition at low Mg2+ concentrations could be attributed to a
chelation of the Mg2+ by ATP. Similar conclusions were reached by
Holmsen and Strom (1969), except that they did note a slight inhibi-
tion by ATP at higher concentration of Mg2+. Their data did not fit
the substrate-phosphate overlap mechanism proposed by Reynard g£_§1.
(1961).

The argument for a chelation mechanism for the ATP inhibition
was countered by Boyer (1969). He showed that the ATP inhibition was
independent of removal of free Mg2+ by ATP. This was done by "buffer-
ing" the free'Mg2+ with glycerol l-phosphate which binds Mg2+'moder-
ately tightly.

The ATP inhibition has been demonstrated for pyruvate kinases

from sources other than muscle (see Tables 1 and 2).

Control of Pyruvate Kinases From Sources Other Than Muscle. "In vivo"
Studies. The total activity of rat liver pyruvate kinase was found

to increase ten-fold with rats fed a high carbohydrate diet over

those with a low carbohydrate diet (Krebs and Eggleston, 1965).
Additional studies showed a twenty-fold increase in yeast pyruvate
kinase grown on a 2 96 glucose medium rather than a 0.6 96 glucose
medium (Hommes, 1966). Both enzymes increased on a high glycerol diet

(Takeda $1.51., 1967; Gancedo g£_§1., 1967). Liver pyruvate kinase

11

decreased in rats made diabetic by alloxan injection. Insulin returned
the level to normal; the return was blocked by either actinomycin or

t _1., 1965).

ethionine (Weber

Dietary regulation of rat liver pyruvate kinase was further
studied by Szepesi and Freedland (1968). They observed that pyruvate
kinase activity was increased by a 90 ab casein diet, but only if the
rats were pre-fed a 90 9b carbohydrate diet for four days. This
increase was inhibited by cycloheximide but not actinomycin. They
concluded that carbohydrate induces the necessary m-RNA formation,
but protein synthesis could be stopped by lack of necessary protein
in the diet. Thus the increase in pyruvate kinase activity with a

high protein diet is via protein synthesis from pre-existing m-RNA.

"In vitro" Studies. Pyruvate kinases from several sources have been
shown to be under metabolic control as well (see Tables 1 and 2).
Moreover, the kinetic properties of these enzymes are consistent
with the allosteric mechanism of enzymatic catalysis (Monod gg‘g1.,
1965).

Plots of enzymatic activity of yeast pyruvate kinase activity
versus the concentration of the substrate or effector, PEP, K+, NHI,
or ADP (at low concentrations of PEP) are sigmoidal, indicating a
cooperativity of binding. The Km of each effector is decreased by
the addition of FDP, which transforms the sigmoid plots into Michaelis-
‘Menten hyperbolic plots (Hess _£'_1,, 1966; Hess and Haeckel, 1967;
Hunsley and Suelter, 1967).

This "feed forward" activation by FDP (Hess g; 31., 1966) has

also been observed in pyruvate kinase from rat liver, but not muscle

12

(Taylor and Bailey, 1967); developing loach embryo (Milman and Yurowitzski,

1967); adipose tissue (Pogson, 1968); Escherichia coli (Maeba and Sanwal,

 

1968); and desert locust fat body, but not flight muscle (Bailey and
Walker, 1969).

Tanaka._£_§1. (1967b) reported the existence of two electrophor-
etically and immunologically distinct species of pyruvate kinase from
rat liver, designated type "M" and "L" (see Table l). The "M" type has
prOperties similar to muscle pyruvate kinase and does not appear to be
under rigid dietary or metabolic control, whereas the "L" type varies
with the diet as described above, follows sigmoid kinetics, and is
activated by FDP. A type "L" pyruvate kinase (determined by immunolog-
ical properties) has been observed in erythrocytes which is not acti-
vated by FDP (Tanaka _E _1., 1967b) suggesting the possibility of two
type "L" enzymes, or two forms of one enzyme. The latter possibility
seems more likely since the potential for FDP activation of liver type
"L" enzyme can be destroyed with incubation (Tanaka 51 a1,, 1967b) or
storage (Susor and Rutter, 1968). Adipose tissue also contains two
interconvertible forms of pyruvate kinase; one sensitive and the other
insensitive to FDP activation (Pogson, 1968).

An abnormality in erythrocyte pyruvate kinase activity has been
observed in the disease hemolytic anemia. This abnormality has been
found to be of two types: 1) low total enzyme activity with a normal
Km for PEP (Valentine gt 91., 1961; Koler g a1, 196k), 2) normal
enzyme activity at saturating concentrations of PEP but a ten-fold

increased Km for PEP (Paglia t al., 1968; Sachs g£_§1,, 1968).

The occurrence of a different, very labile, pyruvate kinase

from tumors has recently been reported (Criss, 1969; Weber, 1969).

13

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LITERATURE REVIEW: PYRIDOXAL CATALYSIS

I have shown that rabbit muscle pyruvate kinase interacts speci-
fically with pyridoxal-5'-phosphate and this interaction results in a
pronounced inactivation (this will be discussed in Chapter 2). It is
of interest to understand the mechanism of this interaction and to
correlate this to the loss of enzymatic activity. In formulating and
evaluating mechanisms for the effects I have found, it is useful to
consider the known mechanisms of pyridoxal-5'-ph03phate interaction
with enzymes per se, and in enzyme catalyzed reactions.

Many aspects of the interactions of pyridoxal-5'-phosphate in
enzyme catalyzed reactions are described below. The interaction with

enzymes per se will be discussed in Chapter 2.

Discovery of Pyridoxal Catalysis. The original experiments which led
to the eventual discovery of pyridoxal catalysis showed that a variety
of lactic acid bacteria required pyridoxine (pyridoxol), an analog of
pyridoxal, for growth on a medium which was determined chemically to
lack it (Moller, 1958; 1959). However, on addition of an animal tis-
sue extract to the pyridoxine deficient medium, the organism grew

umch faster than could be attributed to the pyridoxine content of the
extract. It was concluded that the pyridoxine was converted by the
extract into a metabolite, called "pseudopyridoxine," which was more

active for the bacterial growth than pyridoxine itself (Snell, l9h2a).

The: higher "pseudOpyridoxine" activity of the extract was found to

17

18

result from an interaction of pyridoxine and with the amino acids of
the medium (Snell, 19h2b), and later to the presence of the analogs,
pyridoxal and pyridoxamine (Snell, l9hha, l9hhb).

Pyridoxine derivatives have been shown to be involved in the
decarboxylation of tyrosine in tissue extracts (Gunsalus and Bellamy,
l9hh). Addition of pyridoxal stimulated decarboxylation, but the
addition of ATP, which led to a phosphorylated derivative of pyridoxal,
markedly stimulated the decarboxylation (Gunsalus §£_§1,, 19hh). A
similar stimulation by a phosphorylated derivative of pyridoxal was
shown for the transamination reaction (Schlenk and Snell, l9h5).
This cofactor, named "codecarboxylase" (Gale and Epps, l9hh), was
determined to be the phosphate ester of pyridoxal and was chemically

synthesized (Gunsalus g£_§1., l9h5).

Reactions Catalyzed byiPyridoxal Compounds. Numerous amino acid
reactions have been shown to be catalyzed nonenzymatically by pyri-
doxal plus metal ions or enzymatically by pyridoxal or pyridoxal-5'-
phosphate with no requirement for metal ions (see reviews: Metzler,
._£ _1., 195M; Snell, 1962; Fasella, 1967). These reactions of the
amino acids can be classified into three general groups of reactions

involving cleavage of the three bonds to the abcarbon of the amino

acid. They are shown below with examples for each.

1
Rmégam

NH2

19
1. Labilization of the hydrogen leading to racemization or transamina-
tion:
alanine transaminase: L-alanine +-a-ketoglutarate -———>’ pyruvate
<r-—-
+ L-glutamate
alanine racemase: L-alanine ‘———4‘ D-alanine
11....
2. ardecarboxylation:
histidine decarboxylase: L-histidine --9’ histamine + C02
5. Reaction of the R-Group substituent:
a. Elimination or replacement of the entire group:
threonine aldolase: threonine ___;>. acetaldehyde + glycine
sr--
tryptophanase: RCHECHNHECOOH + R'H ——-> R'CH2C11NH2COOH + RH
tryptOphan + H20 -——4>' indole +-NH3 + pyruvate
h. Modification of the R-group:
cysteine synthetase: serine + H28 '—-€> cysteine + H20

B-aspartate decarboxylase: aspartate '———€> 002 + alanine

General Mechanism for Pyridoxal Catalysis. The first mechanism to
explain pyridoxal catalysis was proposed independently by Braunstein
and Skemyakin (1955) and Metzler _1‘31. (l95h). The mechanism included
the formation of a Schiff base between the amino acid reactant and the
pyridoxal catalyst (shown below). The amino acid was thus assumed to
be "activated" for its subsequent reactions by weakening the bonds of

the a-carbon.

20

R— —COOH

z!
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z—o—m
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o
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o
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c <__> 0H H20 H

The necessity for ketimine formation (compound b, above) as an
intermediate has been postulated but not proven. Alpha-methylserine
which contains no a4hydrogen atom and thus cannot form a ketimine has
been decarboxylated nonenzymatically by PLP plus metal ions (Kalynakar
and Snell, 1962), and degraded to formaldehyde and alanine, nonenzy-
matically and enzymatically (Snell, 1965). Labilization of the a-
hydrogen was shown not to occur during the decarboxylation of tyro-
sine to tyramine by tyrosine decarboxylase (Mandeles ££.él-: 195A);
furthermore, the decarboxylation proceeded with full retention of
configuration (Bellau and Burba, L960).

However, there are many other systems where ketimine formation
clearly occurs. The rate of appearance of the ketimine and the corres-
ponding disappearance of the aldime (compound a, above) has been mea-
sured spectrophotometrically in methanol (Matsushima and Martel, 1967).
The disappearance of a Cotton effect due to a loss of the asymmetric

<Harmer upon ketimine formation has also been shown (Torchinsky and
K0ll'eneva, 1965). Spectral prOperties of the quinoid-like structure of

thEB ketimine have been observed in several enzyme systems (Schirch and

Jenk ins, 19611).

21

Evidence for Schiff base formation between the h-formyl group
of PLP and the amino group of an amino acid is provided by spectral
data. Pyridoxal phOSphate in bicarbonate buffer at pH 7.2 displays
an absorption maximum at 588 mu. The absorption maximum is shifted
to hl5 mp, with the formation of an additional bond at 280 mu, within
ten minutes after addition of an amino acid (Blakely, 1955; Metzler,
1957). The absorbance at h15 mu is attributed to the protonated
Schiff base; the absorption is shifted to 567 mu at higher pH values.

The midpoint for this transition is at pH 10.5 (Metzler, 1957).

NONENZYMATIC PYRODOXAL CATALYSIS. In nonenzymatic catalysis, the
metal ions have two functions: (1) chelation with the 5-hydroxyl
group of the pyridoxal and the imino group of the pyridoxal-amino
acid complex, aiding electron withdrawal (Snell, 1965), (2) main-
tenance of the required molecular planarity (Dunthan, 1966). The
heterocyclic nitrogen atom functions in electron withdrawal, while

the remaining groups appear to be functionless (Metzler 21,51.,

195k).

Enzymatic Pyridoxal Catalysis. The metal ion is replaced by a com-
plex protein molecule in catalysis by pyridoxal in enzyme systems.
Otherwise the same functional group of the pyridoxal molecule are
Operative in the enzymatic catalysis as in the nonenzymatic catalysis.

The formation of a Schiff base with an amino group of an
enzyme was first proposed by Jenkins and Sizer (1957). At pH h.8
tfliey observed an absorption band at h50 mu resulting from the

ilrteraction of pyridoxal-5'-phosphate with glutamic-aspartic trans-

22

aminase. This proposal was given further proof by sodium borohydride
reduction of the enzyme-pyridoxal complexes with subsequent isolation
of pyridoxal-lysine (Matsuo and Greenberg, 1959).

With the pyridoxal cofactor bound to the enzyme via a Schiff
base, an additional step is required in enzymatic, pyridoxal catalysis,
namely "transaldimination"--the transfer of pyridoxal from the enzyme
to the amino acid substrate (Snell, 1962).

The enzymatic mechanism of pyridoxal catalysis is undoubtedly
extremely complex encompassing several factors. The complexity is
shown by spectral data indicating at least four enzyme-pyridoxal
substrate complexes (Schirch and Jenkins, 196R; Jenkins and D'Ari,
1966). These complexes can be attributed to the interaction of
pyridoxal with protein amino acids such as serine, cysteine, and
histidine. Further analysis of these complexes will contribute to

a better understanding of the catalytic mechanism.

CHAPTER ONE

REVERSAL OF THE DISSOCIATION OF RABBIT MUSCLE

PYRUVATE KINASE INTO UNFOLDED SUBUNITS

23

ABSTRACT

The dissociation of tetrameric rabbit muscle pyruvate kinase
(mol wt = 257,000) into unfolded subunits (mol wt = 57,000) in 6 M
guanidine has been shown to be reversible. A systematic study of the
factors affecting the reversal of the dissociation led to conditions
where up to 70 86 of the initial catalytic activity was regained.
The reversal procedure (from: Deal, W. C. (1969), Biochemistry 8,
2795 (1969))consisted of two phases: (1) a 100-fold dilution of
guanidine-dissociated enzyme (00) into a reversal solvent at 00 and
(2) incubation of the resulting solution at a higher temperature,
usually 160. Conditions for optimum reversal of dissociation were
(1) pH 8; (2) protein concentration, 0.0M mg/ml; (5) ionic strength,
0.5 (h) reducing agent, 0.06 M B-mercaptoethanol and (5) temperature--
00 dilution, followed by six hr at 160. The half-time for activity
recovery was approximately #5 min at both 0.02 mg/ml and 0.09 mg/ml
enzyme concentration. Two metabolites, insulin and phosphate ion,
were found to greatly influence the reversal of dissociation. Insulin
decreased the activity recovery upon reversal, in contrast to what
would be expected for an inducer of the enzyme. Ph08phate ion yielded
activity recovery at 56°; neglible activity was recovered at that tem-
perature in its absence. The reversal of dissociation was not affected
significantly by the addition of a number of metabolites including ATP,
ADP, 5'-AMP, 5',5'-AMP, lactate, FDP and NAD+. The reassociated
enzyme had the same Km, heat stability, and sedimentation coefficient

as the native enzyme.

2h

25
INTRODUCTION
We have previously shown (Deal, 1967; Deal and Constantinides,
1967; Deal, 1969) that, contrary to the widely accepted view, some pro-

1 metabolites for 1n_vitro reassembly from

teins apparently require
their unfolded subunits. The experimental evidence was that for 13
21159 reversal of dissociation of yeast glyceraldehyde-5-phOSphate
dehydrogenase, NAfifwas required. It was suggested that the 1g_y1£gg
requirement for NADImay reflect a similar 13.3139 requirement for
NADIfor proper folding of nascent polypeptide chains as they are
being synthesized, thereby providing for control of enzyme synthesis.
The possibility was raised that similar effects might occur with
other enzymes.

As part of an analysis of the metabolic control and structure
of rabbit muscle pyruvate kinase (Steinmetz and Deal, 1966; G. S.
Johnson and W. C. Deal, Jr., in preparation) it was of interest to
determine whether rabbit muscle pyruvate kinase could be reassembled
1g_y1££g_from its unfolded subunits (Steinmetz and Deal, 1966; Kayne,
1966), and if so, whether metabolites were required for, or had any
effect upon, the process.

Pyruvate kinase has often been postulated to be a control point
in glycolysis and the activities of pyruvate kinases from various

t al.,

sources are known to be affected by various compounds (Hess
1966; Milman and Yurowitski, 1967; Tanaka g£_§1., 1967; Taylor and

Bailey, 1967; Maeba and Sanwal, 1968). Also, the synthesis of pyruvate

¥

1This means that in the absence of the metabolite, the rate of
correct folding is extremely slow. The metabolite may, or may not, be
absolutely essential for correctly formed enzyme.

26

kinase from various sources is known to be affected by various condi-
tions. Rat liver pyruvate kinase, but not kidney cortex pyruvate

kinase, decreases upon starvation or low carbohydrate diets and increases
with high carbohydrate diets (Krebs and Eggleston, 1965). This effect
may be explained by the observation that insulin induces pyruvate kinase
synthesis in alloxan diabetic rats (Weber $2.31., 1965; Weber g£_§1.,
1966). This suggested that the synthesis of pyruvate kinase might be
regulated as an important step in metabolic control, and that metabo-
lites or related substances might be the agents which would accomplish
such regulation.

Pyruvate kinase has been well characterized, and its properties
have been discussed in an excellent review by Boyer (1962). Morawiecki
(1960) reported that pyruvate kinase dissociated into subunits of mol
wt 150,000 in 6 M urea and concluded that the enzyme consisted of at
least two polypeptide chains. Our previous work (Steinmetz and Deal,
1966) and that of others (Cottam g£_§1,, 1969) has shown that tetrameric
rabbit muscle pyruvate kinase (mol wt 257,000) is completely dissoci-
ated into unfolded subunits (mol wt 57,000) by high concentrations of
urea (h M’or greater) or guanidine. Optical rotatory dispersion anal-
ysis indicated that the urea denaturation, and presumably the guanidine
denaturation, resulted in essentially complete unfolding of the poly-
peptide chains as well (Kayne, 1966).

Although Morawiecki (1960), using a dialysis procedure, was
able to obtain activity recovery of pyruvate kinase exposed to 2.5 M
urea--which completely inactivates, (Morawiecki, 1960), but only

partially dissociates it (Steinmetz and Deal, l966)--he concluded that

27
the dissociation in 5 M_urea was irreversible, since only 5 96 of the
activity could be regained.

This paper describes a systematic analysis of factors affecting
reversal to determine the Optimal conditions necessary for the renatur-
ation of rabbit muscle pyruvate kinase. Up to 70 56 of the native
activity was regained upon careful dilution of the denatured enzyme
into the proper renaturation solvent. Independently, Cottam _£‘_1.,
(1969) obtained 55-50 96 activity recovery upon removal of the denatur-
ing agent by dialysis or by gel filtration.

Two metabolites were found to influence the reversal of dis-
sociation in a specific way. Insulin inhibited the reversal, in con-
trast to what would be expected for an inducer of the enzyme. The

presence of phosphate ion yielded significant activity recovery at 560;

none was obtained in its absence at that temperature.

RESULTS

Preliminary experiments using the reversal2 solvents and proce-
dures (Deal, 1967; Deal, 1969) for glyceraldehyde-5-phosphate dehydro-
genase immediately gave significant recovery of pyruvate kinase activ-
ity upon appropriate dilution of the guanidine-enzyme solution into
the reversal solvent. A systematic study of variables influencing
the reversal was then conducted, using a successive approximation
technique. All results reported are for experiments where all vari-
ables except that under study were at their optimal values, unless

indicated otherwise.

 

2The words "reversal" and ”renaturation" stand for "reversal
of dissociation or unfolding or inactivation."

28

EFFECT OF pH AND BUFFER SPECIES. Using imidazole, tris, and glycine
buffers (0.05 M), the pH of the reversal solvent was varied from pH
6.2 to pH 9.6. The range for Optimum reversal was found to exist
between pH 7.0 and pH 9.0 (Figure 1). Except for imidazole, all the
buffers tested, including Tris, glycine, bicine and tricine (Good
_1__1,, 1966), bicarbonate, and phosphate, gave essentially identical
recoveries of activity upon reversal at 160 (but see the phosphate
effect at 570). However, at a given pH, the recoveries appeared to
be lower in imidazole. To eliminate the possibility that impurities
in the imidazole might have been interferring with the reversal, the

experiments were repeated using imidazole recrystallized from abso-

lute ethanol; however, this did not improve the recovery.

EFFECT OF SALT SPECIES AND IONIC STRENGTH. An ionic strength Optimum
of 0.2 to 0.5 was found for ammonium sulfate, potassium chloride, and
magnesium chloride (Figure 2). Similar results were obtained with
sodium chloride. The cations of all these salts have been shown to

t al., 1966).

bind pyruvate kinase (Kayne and Suelter, 1965; Suelter
Tetramethylammonium chloride, whose cation does not bind, gave
similar effects. These results suggested that the salt requirement

was solely an ionic strength effect and not salt specific.

EFFECT OF TEMPERATURE AND TIME OF INCUBATION. To determine the Optimal
time and temperature of incubation for reversal, reversal samples were
first diluted at 00 (the first stage of reversal) and then exposed to

various temperatures ranging from 00 to 57°. The samples were assayed

at the indicated times (Figure 5). The results indicated that 12-160

29

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55

was the temperature range for optimal reversal. They also showed that
the activities of the reversed enzyme samples, and those of the controls,
were constant for 2h hr (additional data not shown indicated complete
stability in this system for up to #2 hr)° Maximal reversal was
obtained in 2-5 hr at 210, but about 5 hr exposure was needed for the
120 and 160 samples to attain maximal recovery. Essentially no reversal

occurred in the samples at 00 (not shown).

THE PHOSPHATE EFFECT. The failure to obtain significant reversal under
these conditions at 570 was surprising, since this is the temperature
at which the chains must fold 13’2132. Since a visible precipitate was
observed at the 0.0h mg/ml concentration at 57°, it was thought that a
lower enzyme concentration might lead to less aggregation and a greater
recovery of activity. However, even with concentrations of 0.02 and
0.009 mg/ml in the renaturation solution at 570, no activity was
recovered. Nor did a 50 min preincubation at 00 provide any increase
in recovery.

In an effort to obtain recovery at 570 in Tris buffer, four
other reversal conditions were used: (1) the optimal conditions (see
Methods), with the omission of the 0.h M KCl; (2) the conditions
described in the legend for FigurefS; (5) the conditions in (2) minus
the EDTA, and (h) the conditions in (2) minus the ADP. However, none
gave significant activity recovery.

Other buffers were used and reversal was finally obtained at
560 with phosphate, but not with bicarbonate or imidazole buffers.
Maximum recovery occurred in the range of 0.0h M to 0.1 M phosphate

(Figure h). An unusual characteristic of the phosphate ~56o system

56

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37

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58

was the dependence of activity recovery upon time of preincubation at
00 (see Methods). Preincubation times of 50 sec and 90 min at 00
yielded activity recoveries of 10 96 and 50 86 reSpectively. Denatured
enzyme diluted at 560 directly, instead of at 00, precipitated. In
contrast, denatured enzyme diluted at 160 directly yielded 50 86 activ-
ity recovery with no precipitation. A 5 min preincubation at 00 was

adequate to yield maximal recovery at 160.

EFFECT OF PROTEIN CONCENTRATION. Since some phase or phases of the
reversal process involved the successive association of the subunits
into the dimeric and then tetrameric species, it was expected that
the concentration of enzyme in the reversal mixture might be impor-
tant for activity recovery. To test the effect of enzyme concentra-
tion on the reversal, stock enzyme solution was diluted with the dis-
sociation solvent, the final composition8 of which was 5.5 M
guanidine-HGl, 0.06 M B-mercaptoethanol, 0.02 M_Tris-HC1 buffer

(pH 8.0), 0.07 M KCl, 0.001 M EDTA, plus residual 0.01 y imidazole
buffer (Kayne, 1966). After a one-hr incubation in the dissociation
solvent, varying aliquots of the enzyme solution were diluted
apprOpriately with the dissociation solvent to yield various protein
concentrations. Samples from these solutions were then diluted 100-
fold, to yield reversal samples containing various enzyme concentra-

tions ranging from 0.002 to 0.h mg/ml. Native enzyme controls were

 

3The enzyme was stored in aqueous solution (0.02 M imidazole
buffer) at a concentration of 20-80 mg/ml. The 5.5 M guanidine was
a compromise to allow the highest possible protein concentration in
the dissociation solution with a sufficient guanidine concentration
to insure complete dissociation and unfolding of the enzyme. Since
guanidine usually seems to accomplish the same result as urea at
roughly one-half the concentration, 5.5 M guanidine seemed adequate

t0 insure dissociation and unfolding in this experiment (Steinmetz
and Deal, 1966).

59

not run for this particular series because of the extensive amounts of
enzyme required. The data obtained (Figure 5) indicated that enzyme
concentrations in the narrow range of 0.05 to 0.08 mg/ml were Optimal.
One technical problem preventing analysis of a wider range was that

the enzyme precipitated at protein concentrations greater than 0.2 mg/ml

in the reversal solution.

EFFECT OF SUCROSE. The presence of sucrose in the reversal solution
increased the percent recovery at higher, but not at lower, enzyme
concentrations. Reversal solutions with 10 96 sucrose gave #5 Sb
recovery of activity at 0.12 mg/ml enzyme and 11 86 recovery at 0.2
mg/ml; under otherwise identical conditions, samples without sucrose
yielded 15 9b and 0 96 recovery respectively. However, at a protein
concentration of 0.h mg/ml, no reversal was obtained, even in the
presence of 10 96 sucrose--and a precipitate formed during the incuba-
tion. In contrast to the enhancement by sucrose of reversal recovery
at higher protein concentrations, there was no effect of either 10 96
or 20 96 sucrose on the reversal recovery using a protein concentra-

tion of 0.0M mg/ml, the Optimal enzyme concentration (Figure 5).

HALF-TIMES FOR REVERSAL. Since native pyruvate kinase consists of
tetramers, and also apparently exists as dimers or monomers (5.6 S
species) under certain conditions (Steinmetz and Deal, 1966), it was
of interest to see whether association had to occur in order for
activity to be regained. The concentration dependence of the half-
times of activity recovery at 160 was studied to provide information

on this question. The half-time for recovery, determined experimen-

110

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tally as the time at which 50 96 of the maximum activity was recovered,
was found to be 50 min for both the 0.09 mg/ml and 0.02 mg/ml samples;
the half-time was an min for the 0.0h mg/ml samples (Figure 6). These
differences were presumably not significant, since the two extremes of
concentration had the same half-times. Although these results were not
conclusive, they did suggest that at these concentrations a first-order
process, a folding step, was rate-limiting for the recovery of activity.
The half-time for reversal at 160 was surprisingly long. How-
ever, the half-time for reversal at 560 in phosphate buffer and 0.0h
mg/ml was determined to be only h-5 min (Figure 7). This is closer to

the rate of folding expected 1g_vivo.

EFFECT OFfiREDUCINC AGENT SPECIES AND CONCENTRATION. Since the

reversal experiments were conducted under aerobic conditions, the
possibility existed that random formation of disulfide bonds might

be hindering the specific refolding of the enzyme. To test this
possibility, a reversal study was performed with various concentrations
of the reducing agent B-mercaptoethanol ranging from 0 to 0.hh M,
Maximal reversal was obtained with B~mercaptoethanol concentrations in
the range of 0.05 to 0.15 M,(Figure 8). Negligible recovery was
obtained from the sample reversed without B-mercaptoethanol.

Other reducing agents were also tested. Reversal solutions con-
taining 0.06 M dithiothreitol or 5-mercapto-1,2-propanediol gave results
similar to those with 0.06 M B-mercaptoethanol. Glutathione, which is
expected to exist in fairly high concentrations 13.2132, also gave good
recovery of activity. However, in the glutathione system both the con-

trol enzyme and the reversed enzyme were unstable during incubation,

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owm S. Enema/mm mos mass 5% .E atom:

116

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losing approximately 50 9b of their original activity within ten hr.

EFFEQI 0F ADDED METABOLITES. Previous work in this laboratory had
shown that the refolding of yeast glyceraldehyde-5-phosphate dehydro-
genase was aided by the metabolite NAD+ (Deal, 1967; Deal and
Constantinides, 1967; Deal, 1969). To test for a similar type of
effect on the reversal of pyruvate kinase, reversal solutions 5 my,
in ATP, ADP, 5',5'-AMP, PEP, or lactic acid were tested. All the
metabolites were tested in the presence of 0.1 fl MgClg. Also, 3',5'-
AMP, ADP, and ATP were tested in O.h M_KC1. Occasionally ADP and

ATP in O.h fl KCl appeared to give about 5-10 9b enhancement of activ-
ity recovery over that in their absence, but this was not observed in
0.1 M M8012. None of the other metabolites significantly aided the
reversal under these conditions.

It seemed possible that in this system ionic strength might
have an effect superimposed on metabolite effects, as it had in the
reversal system of yeast glyceraldehyde-5-phosphate dehydrogenase.
The requirement of that enzyme for N'AD+ was essentially absolute at
low ionic strength (0.15 E_KC1) and this requirement was virtually
abolished when the ionic strength was raised to 0.8 (Deal, 1969).
However, reduction of the ROI concentration in the reversal mixture
from 0.1; y; to 0.2 u still did not yield any pronounced metabolite
effects at 16°, using the previously mentioned metabolites and some
additional metabolites (Table 5).

Since phosphate had previously been found to uniquely aid
recovery at 36°, it also seemed possible that it might aid the

recovery at 160 and perhaps produce a synergistic enhancement of

50

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51

reversal with other metabolites. However, it only slightly aided the
reversal at 160 and there was no significant effect of added metabo-
lites in the presence of phosphate (Table 3).

Insulin, an inducer of rat liver pyruvate kinase (Weber _£._l.,
1965), showed a pronounced interference with reversal at 160 and an
even more pronounced interference at 570 (Table h). Insulin had no
effect on the native enzyme. Ribonuclease and bovine serum albumin
at the same concentration (0.2 mg/ml to 0.01 mg/ml) had no signifi-
cant effect on the reversal recovery of pyruvate kinase, suggesting

that the effect was specific for insulin and not a general protein

effect.

COMPARISON OF NATIVE AND REVERSED ENZYME. The native and reversed
enzymes were found to have identical characteristics as shown by
measurements of sedimentation coefficients (Figure 9), Km values for
ADP (Figure 10) and K+ (Figure 11), and heat stability profiles at

500 (Figure 12).

DISCUSSION

The systematic analysis of variables influencing the refolding
and reassembly of guanidine-dissociated and unfolded rabbit muscle
pyruvate kinase into its active native tetrameric structure led to
conditions where substantial activity recovery (up to 70 9b) was
obtained. In contrast Morawiecki (1960) had earlier reported the
dissociation in high concentrations of urea to be essentially
irreversible, since he had obtained less than 5 9o recovery of activ-

ity. Also in limited studies Cottam _£ 21.- (1969) obtained 35-50 °/o

52

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n

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56

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61

recovery of activity upon removal of the denaturing agent by dialysis
or gel filtration. Thus, the yeast glyceraldehyde-3-phosphate dehydro-
genase reversal conditions and procedures (Deal, 1967; Deal, 1969)
which served as the starting point for this study, have been found to
give substantial recovery of activity for rabbit muscle pyruvate
kinase, as they have for a number of other enzymes (see Deal, 1969).
As might be expected, the general optimum requirements for
reversal of rabbit muscle pyruvate kinase are similar to those of
these other enzymes. For the yeast glyceraldehyde-3-phosphate
dehydrogenase reversal system and for pyruvate kinase in particular,
two key factors in obtaining significant reversal were: (1) the
removal of the denaturing agent by dilution of the guanidine-enzyme
(0°) into a reversal solvent at 0° and then incubating the sample to
higher temperatures (12-550) to produce the refolding-reassociation
reactions and (2) the use of low protein concentrations, about 0.05
mg/ml, for the reversal process. Both of these factors seemed to
operate to avoid nonspecific aggregations which would have caused

the protein to precipitate, or irreversibly aggregate.

POSSIBLE EFFECTS OF METABOLITES. When this research was begun, the
possibility seemed good that metabolites might affect the folding and
synthesis of pyruvate kinase and/or its activity. Because of the
possible direct relationship presumed (Deal, 1969) between folding
control by metabolites ig’zi££2_and regulation of protein synthesis
by inducers $3,2112, it was natural to consider as prime candidates
for "folding control" those compounds thought likely to be inducers

(or repressors) of pyruvate kinase. In this connection the report by

62

Weber and co-workers (Weber gt al., 1965; Weber 3; al., 1966) that
insulin is an inducer of rat liver pyruvate kinase was of special
interest. This raised the possibility that insulin, or some product
of insulin action, might control the folding of pyruvate kinase.
Unfortunately, the primary products of insulin action have not yet
been elucidated. Therefore, within this group, the only feasible
test was that of the ability of insulin to directly enhance reversal.

This study showed that under the conditions used, insulin did
affect the reassembly of pyruvate kinase, albeit in a negative way
rather than the positive way expected. It also showed that the bind-
ing was specific for insulin; neither bovine serum albumin nor ribo-
nuclease had any effect on the reversal recovery. Furthermore, the
insulin effect was not a nonspecific inhibition of enzyme activity,
since the activity of the native enzyme was not significantly affected
by insulin under identical conditions. Since insulin does interact
with the unfolded polypeptide chains of pyruvate kinase, and since
other variables may superimpose their effects onto those of insulin
in_yixg, the possibility cannot be ruled out that the insulin inter-
action might favor assembly ig_yiyg, rather than impede it.

It also seemed reasonable that the metabolites expected to
influence enzyme activity might be expected to be prime candidates
for affecting the rates of synthesis of the enzyme. A number of com-
pounds have been shown to affect the activity of muscle pyruvate
kinase. The ion, K+, was reported to be required for the enzyme to
be in the proper conformation for activity (Melchior, 1965). The

binding of substrates and activating cations was reported to have

65

resulted in a change in protein conformation (Kayne and Suelter, 1965).
Other studies showed an interaction of the Mg2+eADP complex with the
enzyme (Melchior, 1965). Weber _Eflgl. (1967) showed that acetyl CoA
was an inhibitor of both liver and rat muscle pyruvate kinase.

Also, a number of compounds have been shown to affect the activ-
ity of the pyruvate kinase enzymes obtained from various sources other
than muscle. Pyruvate kinase from E, £011 was activated by both AMP
and FDP (Maeba and Sanwal, 1968) and the enzyme from deve10ping loach
embryos was activated by 5',5'-AMP and FDP (Milman and Yurowitzki,
1967). The pyruvate kinases from yeast (Hess gg‘gl., 1966) and liver
(Taylor and Bailey, 1967) were strongly activated by FDP. Although
there has been no evidence that pyruvate kinase from rabbit muscle
was activated by FDP, Taylor and Bailey (1967) have suggested that
in zi!2_it might have possessed this characteristic and lost it during
isolation and purification of the enzyme.

Of the metabolites and ions tested in this study, only phosphate
and insulin affected the in_yi££2_folding and assembly of pyruvate
kinase from its unfolded subunits. PhOSphate seemed to be absolutely
required for this process at 360, although not at 160. Since this is
near the temperature at which the in yizg_folding of rabbit muscle
pyruvate kinase occurs, this may be a very significant requirement.

In this regard, it is of interest that the rabbit muscle
pyruvate kinase apparently can exist in two temperature-dependent
states (Kayne and Suelter, 1968). Since the midpoint of the transi-
tion from one state to the other is in the range of 160-220, it is

possible and even likely that the low temperature form, which does

6h

not require phosphate for folding, may not occur 33.2113. That is,
possibly only the high temperature form occurs in_gigg and it may
require phosphate for folding. However, an alternative possibility is
that phosphate keeps the enzyme in the low temperature form which
reverses easily. Also, the possibility cannot be overlooked that in
3332 other effects might Operate in conjunction with the phosphate
effect. These subjects are receiving further study.

In general, the muscle is expected to have a considerably dif-
ferent set of metabolic priorities and types of controls than liver
or yeast because it does not have the ability to carry out gluco-
neogenesis, nor does it have, in significant amounts, many of the
other metabolic pathways (such as the pentose phosphate cycle) which
liver and yeast possess. It will thus be of much interest to compare
these results for rabbit muscle pyruvate kinase with pyruvate kinases

from other sources.

MATERIALS AND METHODS

ENZYME PREPARATION AND ASSAY. Pyruvate kinase (EC 2.7.1.I+o) was iso-
lated from frozen rabbit muscle (Pel Freez Biologicals, Inc.) using

the modifications (Steinmetz and Deal, 1966) of the method of Tietz

and Ochoa (1958). The enzyme was assayed at 250 by coupling the
pyruvate kinase reaction to the lactic dehydrogenase reaction and
following the decrease in absorbency at 3&0 mu (Kubowitz and Ott, l9hh)
using a Beckman DU spectrophotometer attached to a Gilford multiple
sample absorbance recorder. Enzyme concentrations were determined by

measuring the Optical density at 280 mu and using the extinction coef-

65

-1 ~l ..
ficient ofi€280 mu = 0.5M m1 mg cm (Bucher and Pfleiderer, 1955).
The specific activity of the native enzyme was found to be 1h0-160
umoles of NADH consumed/min/mg protein. The enzyme concentration in

the assay was 0.2 ug/ml and the reaction was monitored for 1 min.

REAGENTS. Reagent grade chemicals were obtained from the following
commercial sources: Tris (Trizma base), PEP (tricyclohexylamine salt),
glucose-6-phosphate (disodium slat), bovine serum albumin, bovine
pancreas ribonuclease, AMP (sodium salt), bovine pancreas insulin, FDP
(sodium salt), 5',5'-AMP (sodium salt), and lactic acid from Sigma

(St. Louis); ATP (sodium salt), ADP (sodium salt), fi-NADH (disodium
salt), and B-NAD+ (disodium salt) from P-L Biochemicals (Milwaukee);

tricine and bicine (Good t al., 1966) and dithiothreitol from

CalBiochem; imidazole and tetramethylammonium chloride from Eastman
(Rochester, N.Y.); glycine from General Biochemicals (Chagrin Falls,
Ohio); 5-mercapto-1,2-pr0panediol from Aldrich Chemical Co.
(Milwaukee); B-mercaptoethanol from Matheson Scientific (Elk Grove
Village, Illinois); abD-glucose from Pfanstiehl Laboratories, Inc.
(Waukegan, Illinois); and beef heart lactic dehydrogenase from
Worthington (Freehold, New Jersey).

Urea (Baker Analytical Reagent) was recrystallized from
absolute ethanol and allowed to dry at 500 to remove residual ethanol.
Guanidine-HCl was prepared (Anson, l9hl) from Guanidine carbonate
(Eastman, Rochester, N.Y.), and recrystallized from absolute ethanol.

The crystals were then dried at 500. All pH measurements and buffer

0
adjustments were made at 2% .

66

DISSOCIATION PROCEDURE. Unless otherwise indicated, the denaturation
in all experiments was conducted in the following manner: stock
enzyme solution (20 mg/ml, 0.02 imidazole (pH 7.0), 0.001 M_EDTA) was
diluted to h mg/ml in freshly prepared dissociation solution and
allowed to remain there for one hr in an ice bath. The dissociation
solution was at pH 8 and consisted of 7 M_guanidine~HCl or 7 M urea,
0.12 y; B-mercaptoethanol, 0.01. g Tris-H01 and 0.001 3 EDTA.

Activity recoveries using urea or guanidine were identical.
The buffer species or pH of dissociation were not important since
dissociation at pH 6.5 (potassium phosphate), pH 7.5 (imidazole), and
pH 8.0 (Tris) all gave essentially the same activity recoveries upon
reversal. Also, omission of EDTA or B-mercaptoethanol from the dis-
sociation did not affect the reversal recovery. The use of a 100-fold
dilution of urea- or guanidine-enzyme, which resulted in only 0.06 M
residual denaturing agent, yielded high recoveries. This level of
residual denaturing agent had no effect on the activity of native
control samples. Although this does not exclude the possibility that
the level of residual denaturing agent might interfere with the sub~

unit refolding process, it does seem unlikely.

REYERSA; PROCEDURE. The renaturation conditions and procedures followed
were those previously described for the renaturation of glyceraldehyde-
5-phosphate dehydrogenase (Deal, 1967; Deal and Constantinides, 1967;
Deal, 1969). The Optimal conditions for the reversal were: (1) pH 8,
0.05 31 Tris-HCl, (2) protein concentration, 0.0h mg/ml, (5) salt, 0.1+ _rg
KCl plus 0.1 M MgCla, (h) reducing agent, 0.06 M_B-mercaptoethanol, and

(5) temperature-~0o dilution followed by six hr at 160. These Optimal
1

67

conditions were used for reversal except where designated in the apprOp-
riate legends. The dissociated enzyme was diluted with careful swirling
into the reversal solution which had been previously cooled in an ice
bath to approximately 00. The samples were then taken to 160 for incu-
bation. Although in some cases the conditions used varied slightly,
they remained in the Optimal range plateau in all cases. The reducing
agent was added to the stock reversal solvent just prior to the protein
dilution to prevent undesirable air oxidation of the sulfhydryl groups.
In each experiment, control samples were subjected to identical treat-
ment except for the presence of urea or guanidine. Duplicate samples

were usually run.

_QOMPARISON STUDIES. The sucrose density centrifugation experiment fol-

 

lowed the procedure of Martin and Ames (1961). The Spinco Model L
ultracentrifuge was used and the SW-59 rotor was run at h0,000 rpm at
20 for 18 hr.

For the heat stability and LineweaverwBurk analyses, the
reversed and native enzymes (0.0h mg/ml) in 0.05 M Tris (pH 8.0), 0.3 M
KCl, 0.1 M MgClg, and 0.06 M B-mercaptoethanol were dialyzed 12 hr
against 50 volumes of 0.05 M Tris, pH 8.0.

The half-time for the inactivation at 500 under these conditions
was about 18 min. This temperature appears to be near the transition

point, since at M20 there was only 25 86 activity loss in one hr.

CHAPTER TWO

INACTIVATION OF TETRAMERIC RABBIT MUSCLE PYRUVATE KINASE

BY SPECIFIC BINDING OF 2-h MOLES 0F PYRIDOXAL-5'-PHOSPHATE

68

 

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69

ABSTRACT

Rabbit muscle pyruvate kinase is 90 9b inactivated by binding
2-h moles of pyridoxal-5'-phosphate (PLP) per mole of tetrameric enzyme.
Incubation with PLP in 0.2 M imidazole (pl-I 7.5) at 25° results in a
time-dependent loss of enzymatic activity which reaches a final value
in 10-20 min at 250. Half maximal loss of activity occurs with 0.0h
mM PLP. The inactivation does not cause a gross conformational change.
The inactivation is first order with respect to PLP concentration and
enzyme concentration; the second order rate constant is 57 Mfl sec"l
Increasing ionic strength decreases the rate of inactivation by PLP
but low concentrations (1-10 mM) of divalent cations increase it above
the level with no salt. The phosphate containing metabolites, ADP,
ATP, phosphoenolpyruvate, and fructose-diphosphate also decrease the
rate; the effect is more pronounced with low concentrations (0.1 mM)
of divalent cations. The inactivation is relatively specific for PLP
since various analogues including pyridoxamine, pyridoxamine-S-phosphate,
and pyridoxal cause little or no inactivation. Reduction with NaBH4 at
various concentrations of PLP shows that there are 2 types of binding:
(1) a specific, inactivating binding, involving 2-h moles PLP bound
per mole enzyme, and (2) a nonspecific, noninactivating binding which
can involve up to about 20 additional moles PLP bound per mole enzyme.
Both types involve Schiff base formation with E-NH2 groups of lysine.

Reversal of the inactivation is accomplished, only with unreduced

enzyme, by dilution, by dialysis, or by addition of Tris.

70

INTRODUCTION

Pyridoxal-5'-ph03phate (PLP) is required for many enzymatically
catalyzed reactions, such as transamination and decarboxylation, in
which it participates through formation of a Schiff base. (For a
review, see (Fasella, 1967)). In addition, PLP has a different func-
tion in several enzyme systems. It is required for the prOper quater-
nary structure of tryptophanase (Morino and Snell, 1967) and L-
aspartate B-decarboxylase (Tate and Meister, 1969). Also PLP is
required for glycogen phosphorylase activity but reduction of the
PLP-enzyme complex with sodium borohydride does not alter the
enzymatic activity (Fisher g£_§l,, 1958).

Aside from its normal functions in enzyme catalysis, PLP, with
its active aldehyde group, is an ideal reagent for chemical modifica-
tion of enzymes as a means of identifying and studying enzyme func-
tional groups involved in catalysis. For example, through formation
of a Schiff base with €¥amino groups of lysine residues, it inactivates
the following enzymes: glutamic dehydrogenase (Anderson $5.31., 1966),

hexokinase (Grillo, 1968), 6-phosphogluconate dehydrogenase (Rippa

t al.,

__£._l., 1967), and fructose-1,6-diphosphate aldolase (Shapiro
1968). It also inactivates adenylic acid deaminase (Kaldor and Weinbach,
1966) but the mechanism for inactivation has not been determined. With
aldolase and 6-phosphogluconate dehydrogenase, PLP is thought to bind
specifically to the same site as the phOSphate of the substrate. It
also binds to bovine plasma albumin (Dempsey and Christensen, 1962),

through at least three types of binding sites, all involving that-amino

groups of lysines. Treatment of fructose-l,6-diphosphatase with PLP

71

followed by reduction with sodium borohydride yields an active pyridox-
amine-phosphate derivative of the enzyme which is no longer sensitive
to allosteric AMP inhibition (Marcus and Hubert, 1968; Krulwich 95 al.,

1969). PLP lowers the oxygen affinity of hemoglobin (Benesch gglgl.,
1969) .

In the course Of a study of the effects of metabolites upon the
reassembly of rabbit muscle pyruvate kinase (E.C. 2.7.l.h0) from
unfolded subunits (Johnson 23 gl,, 1969), we discovered that pyruvate
kinase was inactivated in the presence of PLP. Since little was known
about the functional groups involved in the catalytic mechanism of
pyruvate kinase, we undertook a detailed study of this effect to learn
more about the mechanism of catalysis as well as to evaluate this
effect as a possible mechanism for in_gigg control of the enzyme.

We present here a detailed analysis of the inactivation of
pyruvate kinase by PLP. Evidence is presented for a PLP binding to
2-h specific lysine groups. The results also provide further infor-
mation on the roles of substrates and divalent cations in the reaction
mechanism. A preliminary report of this research has been published

(Johnson and Deal, 1969).

RESULTS
Incubation of rabbit muscle pyruvate kinase with various con-
centrations of PLP in 0.2 M imidazole (pH 7.5) at 250 resulted in a
time-dependent loss of enzymatic activity which reached a final value
within 10-20 min (Figure 15). NO further change occurred with con-
tinued incubation up to 7 hr, so it was assumed that these represented

equilibrium values. The midpoint or 50 Sb point for the inactivation

 

 

72

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75

Figure 15

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transition was determined to be 0.0h mM from a graph Of equilibrium
activity values as a function of PLP concentrations (Figure 1h).
Inhibition was most pronounced at pH 7.5 (Figure 15) so this pH was
chosen for the detailed analysis.

The inhibition was relatively specific for PLP. Incubation for
50 min at 250 with 1 mM pyridoxal caused only a 20 96 loss of activity
(see Table 5 for additional data). Similar treatments with 5 mM,
pyridoxamine, 5 mM pyridoxamine-S'-phosphate, 0.1 mM acetaldehyde, or
0.1 mM benzaldehyde had no effect on the activity.

Additional experiments were performed to check directly for
instantaneous inhibition of pyruvate kinase activity by pyridoxal com-
pounds during the assay. In direct spectrOphotometric assays, which
couple the reactions of pyruvate kinase and lactate dehydrogenase, an
apparent inhibition was observed with PLP, pyridoxamine, or pyridox-
amine-5'-phosphate, but not with pyridoxal. The apparent Ki’ about
1 mg; was not due to inhibition of pyruvate kinase, since this effect
was not Observed in the direct measurement of pyruvate kinase activity
using the pH stat. Direct assays of lactate dehydrogenase in the
presence of these compounds confirmed that the interference was with

this reaction.

‘SPMQTRAL ANALYSIS. The spectrum of the mixture of PLP and pyruvate
kinase was consistent with that expected for the formation of a Schiff
base between PLP and the é-NHE group of lysine residues (Shaprio _£_§l.,
1968) showing absorption bands at #52 mp and 5h2 mu (Figure 16). The
162 mp absorbance has been attributed to the presence of a protonated

Schiff base and the 5M2 mu absorbance to an unsubstituted aldimine

 

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76

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76

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81

(Metzler, 1957; Snell, 1958). The PLP-pyruvate kinase complex reduced
with NaBH4 had an absorption maximum (Figure 16) at 525 mu as expected

(Fischer gt El-1 1959; Matsuo and Greenberg, 1959).

REDUCTION WITH SODIUM BOROHYDRIDE. Reducation of the pyruvate kinase-
PLP complexes at various concentrations of PLP yielded covalent com-
plexes with different ratios of moles PLP bound per mole enzyme (Figure
17). The biphasic character of the curve suggested two different types
of binding sites: (a) a strongly binding, inactivating type and (b) a
weakly binding; noninactivating type. It appeared that there were
about h strongly binding sites and about 20 or more weakly binding
sites. The data show that up to 25 moles of PLP were bound per mole

of protein. However, since amino acid analysis (Kayne, 1966; Cottam
g£__l., 1969) indicates the rabbit muscle pyruvate kinase contains
about 150-160 lysine residues, it is likely that considerably more

PLP would be bound at higher PLP concentrations.

The data (Figure 17) also show that PLP binding at the strongly
binding sites accounted for almost all of the inactivation of the
enzyme and that PLP binding at the weakly binding sites did not seem
to inactivate the enzyme; it retained 10 96 residual activity, even
with 25 moles PLP bound per mole enzyme. This suggested that the
strongly-binding, inactivating sites may be specific and that the
weakly binding, noninactivating sites may be non-specific, since they
include as many as 25 of the approximately 150-160 remaining potential
binding sites (Kayne, 1966; Cottam gg‘al., 1969).

However, the data in Table 5 showing that l.h moles PLP per

mole enzyme could be bound with only 5 96 inactivation, not the pre-

82

 

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85

Figure 17

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TABLE 5. SODIUM BOROHYDRIDE REDUCTION OF COMPLEXES OF PYRUVATE KINASE
AND PYRIDOXAL COMPOUNDS

The solutions containing pyruvate kinase (0.59 mg per ml) in 0.2 M_imid-
azole, pH 7.5, were incubated for 50 min at 25 with the indicated con-
centration of inactivation compound. The complexes of pyruvate kinase

and pyridoxal comgound were reduced with sodium borohydride and dialyzed
for 18-2h hr at h .

 

 

 

Compound Present Moles Comp. red./Mole prot. 86 Act. Remaining
None --- 100

0.2 mM_pyridoxal-

5'-Phosphate h.7 17

0.8 mM pyridoxal-

5'-Phosphatea l.h 95

0.8 mM pyridoxal 1.6 96

h.0 mM pyridoxal 5.5 70

2.0 mM pyridoxal
then, 0.2 mM pyri-
doxal-5'-Phosphate --- 17

 

a
No 50 min incubation prior to the reduction.

b50 min incubation with pyridoxal, then 50 min incubation with
PLP.

85

dicted 50 20, indicated that the distinction between types of binding
sites was not absolute. Thus, as many as 2 of the h strongly binding
sites may be noninactivating.

The specific (strongly binding) and nonSpecific (weakly binding)
sites were not distinguishable by their spectral properties, since com-
plexes with 5.6 or 12 moles of PLP bound per mole of enzyme (Figure 17)
had the same absorption maximum (525 mg) and had no apparent differ-
ences in the shapes of their curves except for the increased extinc-
tion due to increased numbers of PLP bound.

Enzyme complexes with pyridoxal were also reduced with sodium
borohydride (Table 5). These results show that pyridoxal did bind and
inactivate but much less effectively than PLP. Sodium borohydride

had no effect on the activity of the native enzyme.

RATE OF INACTIVATION. Since the rate of pyruvate kinase inactivation
was dependent upon the concentration of PLP employed (Figure 15),
experiments were conducted to determine the order of the reaction.
Logarithmic plots of residual activity versus time (Figure 18) were
linear, indicating that the reaction was first-order with respect to
enzyme concentration. In addition, the rate of inactivation was
directly proportional to PLP with the four concentrations tested
(Figure 19), showing that the inactivation was first-order with
respect to PLP concentration.

However, increasing the concentration to 0.5 mM did not increase
the rate of inactivation prOportionally (Figure 19). But this point is
subject to large experimental error, as can be seen from the very steep

curve at this concentration (Figure 15).

86

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87

Figure 18

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Since the rate of inactivation can be expressed as

dA
dt

 

Eqn (1) - = k. (PLP) (A)

where A = activity = enzyme concentration, and k2 is the second-order

rate constant, then

Eqn (2) - {—3— = k. (PLP) (A) = to" (A)

where
Eqn (5) kip = k2 (PLP)

p is a pseudo first-order rate constant. Values of klp as a

and kl
function of PLP concentration were calculated from the data in Figure
18. From Eqn (5), the plot of the pseudo first-order rate constant,
klp, vs PLP concentration should have a slope equal to k2, the second-
order rate constant. From such a plot (Figure 19) the second-order
rate constant was found to be k2 = 57 Mil sec-l

Since most of the activity of pyruvate kinase was lost upon
binding only h moles PLP per mole enzyme, it seemed possible that PLP
was binding at or near the active site. Thus it appeared that a sub-
strate or a required cation, or another metabolite might affect the
inactivation. Accordingly, several such compounds were tested. The
species of salt used and certain metabolites were found to affect the
rate of inactivation (Figure 20, Figure 21, and Table 6), but not the

equilibrium (final) activity values. All the equilibrium values were

within 10 96 of that with no added salts or metabolites.

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TABLE 6. EFFECT OF METABOLITES ON THE RATE CONSTANTS FOR INACTI-
VATION OF PYRUVATE KINASE BY PLP

Conditions as in Figure 8.

 

Contents Rate Constant (sec-1)

 

Metabolites Plus No Added Cations

0.2 M imidazole 0.0075
+ 1 mM ADP 0.0061
+ 1 mM ATP 0.0061
+ 1 mM P-Enolpyruvate 0.0055
+ 1 mM pyruvate 0.0075
+ 1 mM Fructose-l, 6-diphosphate 0.0068

Metabolites Plus Added Monovalent Cations

0.2 M imidazole + 50 mM KCl 0.0055
+-1 mM ADP 0.0050
+ l mMP -Enolpyruvate 0.0050
+ 1 mM Fructose-1,6-diphosphate 0.0056

Metabolites Plus Adde_d__2ivalent Cations

0.2 M imidazole + 1 mM P-Enolpyruvate 0.0055
+ O. 08 mM MgClp ' 0.00h8
+ 0.25 mM MgCl; 0.00115
+ 0.5 mM MgCl. 0.00145
+ 1.0 mM MnCle 0.0010
+ 5 0 mM MnCle 0.001-L5
+10 0 mM MnClp 0.0058
+ 1.0 mM CaClo 0.0015
0.2 M imidazole + 1 mM ADP 0.0061
+0.12 mMMnCle 0.0060
+ 0.25 mM Mn012 0.0060
+ 5.0 mM MnCla 0.0100

Metabolites Plus Added Monovalent and Divalent Cations

0.2 M imidazole + 50 mM KCl + 5 mM MnClp 0.0075
+ 1 mM ADP 0.0056
+ 1 mM ATP 0.0056
+ 1 mM P-Enolpyruvate 0.0060
+ 1 mM pyruvate 0.0075
+ 1 mM Fructose-1,6-diphosphate 0.0066

 

96

The control samples without PLP were relatively stable, losing
only about 5 96 activity per hr. The sample containing 1 mM fructose-l,
6-diphosphate was an exception to this; it lost 20 96 activity per hr.

At a constant PLP concentration (0.2 mM) the rate of inactivation
(determined from Figure 22 and similar plots) was dependent upon salt
species and salt concentration. The pseudo first-order rate constant
decreased monotonically with increasing concentrations of monovalent
cations (Figure 20) but with increasing concentrations of divalent
cations it reached a maximum and then decreased (Figure 21).

The decrease in rate constant with increasing monovalent cation
concentrations appears to be an ionic strength effect since the activat-
ing salts, potassium chloride and potassium sulfate (Boyer, 1962) show
equivalent effects at equal ionic strengths (Figure 20). The less
effective activating salt, sodium chloride, and the nonactivating salt,
tetramethylammonium chloride, show an even greater protection than
potassium chloride.

The divalent cation effect (Figure 21) was observed with acti-
vating cations (Mg+2 and Mn+2), as well as with the inhibitory cation,
Ca+2 (Boyer, 1962).

The metabolites ADP, ATP, phosphoenolpyruvate, and, to a slight
extent, fructose-1,6-diphosphate protected the enzyme against inactiva-
tion by PLP (Table 6). In contrast, 1 mM pyruvic acid, AMP, 5',5'-
cyclic AMP, fructose-6-phosphate, NAD+, or inorganic phosphate, or
insulin (0.2 mg per ml) did not protect. There was a large decrease
in the rate of inactivation with phOSphoenolpyruvate and ADP in the

presence of MnC12 or MgCl2 (Figure 22, Table 6). This effect was seen

 

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98

Figure 22

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99

with ADP only at lower concentrations of divalent cations. This is in
contrast to the increased rate of inactivation caused by divalent
cations alone.

A solution of partially inactivated (in 0.2 mM PLP) pyruvate
kinase with approximately 3 moles of PLP covalently bound per mole of
protein was further inactivated in 0.2 mM PLP. It was found to be
inactivated at the same rate and to reach the same final 96 inactiva-

tion as unreduced enzyme.

STRUCTURAL ANALYSIS. The enzyme inactivated by PLP was centrifuged

in a sucrose density gradient containing PLP. The otherwise inactive
enzyme was located by reversing the inactivation by addition of excess
Tris to each fraction. No gross conformational change accompanied the
inactivation since native enzyme and inactivated enzyme showed identi-

cal sedimentation prOperties (Figure 25).

REVERSAL OF THE INACTIVATION. Complete recovery of activity was
obtained by three methods: (a) by a h0-fold dilution of the PLP inac-
tivated enzyme with buffer; (b) by a 16 hr dialysis at ho; or (c) by
addition of 0.05 M Tris buffer, which completed with the protein amino

groups to form a Schiff base with PLP.

DISCUSSION

POSSTELE BIOLOGICAL SIGNIFICAMQE, There are only a few known inhibi-
tors of muscle pyruvate kinase; moreover, none of these has been sug-
gested to be a potential agent for metabolic control ig_yiyg. This
study shows that under the conditions used in this study, rabbit

-5
muscle pyruvate kinase is 50 96 inactivated by h x 10 M_PLP. The

 

100

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101

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(1W/Slan) - All/“13V

102

concentration of PLP in muscle (rat) is h.65 x 10.6 M (Wachstein and
Moore, 1958; Long, 1961), and the concentration of free PLP may be even

lower due to specific binding of PLP to other enzymes.

NUMBER OF BINDING SITES. Pyruvate kinase (mol wt 257,000 has been shown
to be composed of four subunits of mol wt 57,000 (Steinmetz and Deal,

1966) which by several tests appear to be highly similar (Cottam t al.,

1969). This raises the possibility that there might be four active
sites per tetramer; however, only two have been found in previous
studies (Reynard _£._l,, 1961; Mildvan and Cohn, 1965). The results
reported in this study do not relate directly to the number of catalytic
sites in the enzyme. The data do suggest that there are 2 to h unusually
reactive lysines. Furthermore it is possible that these are near
active sites, since the characteristics of PLP binding to these unique
lysine groups are so strongly and specifically influenced by the cations
and substrates of the pyruvate kinase reaction. However, they do not
appear to be part of the active site (see later section on Mechanism of
Inactivation).

There are at least two possible explanations for the fact that
the enzyme is not completely inactivated: (a) the binding by PLP
renders the active sites only 10 ab active or (b) an equilibrium exists
between enzyme with PLP bound and enzyme without PLP bound.

In the first explanation, the first four PLP molecules bound
convert the tetrameric enzyme to a species which is only 10 96 active.
The remaining PLP molecules bind at noninactivating sites and cause

only negligible further loss of activity. This explanation agrees best

with the reduction data.

105

The second explanation, which may be called the equilibrium mix-
ture explanation, assumes that when PLP is bound to a specific binding
site, it completely inactivates that site. Hence, an equilibrium is
formed between free enzyme sites, inactivating sites with PLP bound,
and noninactivating sites with PLP bound. The residual activity is
presumed to come from the free enzyme sites or enzyme with only nonin-
activating sites bound.

This second explanation would appear to be ruled out by the data.
Increasing the PLP concentration should drive the reaction completely
to products, yielding complete inactivation. This was not observed;
the inactivation seemed to reach a plateau at the high PLP concentra-
tions used. However, there is one possibility which would be consis-
tent with both the second explanation and the data. This involves
postulating that the initial binding of 2-h PLP molecules would lead
to some unfolding of the enzyme, thereby exposing previously inacces-
sible, noninactivating sites. These newly available, noninactivating
sites could then compete with inactivating sites for PLP and greatly
increase the amount of PLP required for inactivation over that expected
from the results during the initial stages of binding. In this case
the overall results would be the sum of two or more equilibria, not
one. The highest PLP concentrations used in these studies led to

binding of only about 25 96 of the total 150-160 lysines.

MECHANISM OF THE INACTIVATION. One question is whether PLP inactivates
rabbit muscle pyruvate kinase by binding at or near the active site, or
whether it binds elsewhere and inactivates the enzyme by a conforma-

tional change. There is no evidence for instantaneous inhibition of

10h

the enzyme by PLP; all the curves of activation as a function of time
extrapolate back to maximal activity at 0 time.

Since there is no instantaneous inhibition by PLP, it does not
seem likely that PLP binds at the phosphate binding site of the sub-
strates. This is further supported by the lack of pronounced protec-
tion by substrates against inactivation under equilibrium conditions.
It is interesting that the phosphate-containing substrates, ADP, ATP,
and P-enolpyruvate (but not pyruvate) decrease the rate of inactiva-
tion of the enzyme by PLP. It appears that they do this not by bind-
ing at the active site, but by stabilizing the native conformation,
thereby hindering the conformational changes required for inactivation.
The inactivation of rabbit muscle pyruvate kinase by binding of PLP to
specific sites that are not catalytic sites differs from that found
with certain other enzymes. PLP inhibits aldolase (Shapiro 33 al.,
1968) and 6-phosphogluconate dehydrogenase (Rippa gt gl,, 1967) by
binding at the same site as the phosphate 0f the substrate.

The PLP data indicate that divalent and monovalent cations
have completely different effects on the inactivation. The rate of
inactivation decreases monotonically with increasing monovalent cation
concentration (Figure 20) but it goes through a maximum with increas-
ing concentration of divalent cation (Figure 21). In contrast to this,
there is a decrease in the rate of inactivation with low concentrations
of divalent cation in the presence of the substrates ADP or phospho-
enolpyruvate. The monovalent cations and higher concentrations of di-
valent cations apparently retard the inactivation by stabilizing the
native enzyme conformation via a relatively nonspecific ionic strength

effect.

105

There are three general possible roles which metal ions may play
in the PLP inactivation in the presence and absence of substrates:
(a) they may cause a conformational change in the enzyme, (b) they may
complex with PLP, (c) they may complex with the substrates. These
possibilities have known counterparts in the mechanism of action of PLP
and the mechanism of pyruvate kinase catalysis: (a) The monovalent
cations are thought to provide the enzyme conformation necessary for
catalysis (Melchior, 1965). Also, divalent cations bind to the enzyme
(Mildvan and Cohn, 1965) and cause conformational changes in it
(Suelter and Melander, 1965). Such conformational changes have been
postulated to explain the protection by Mn+2 against inactivation of
pyruvate kinase by p-mercuribenzoate (Mildvan and Leigh, 196%).
(b) A chelation of divalent cations by PLP has been postulated in the
nonenzymatic catalysis by PLP (Metzler and Snell. 1952). (c) Both
phosphoenolpyruvate and ADP are known to bind divalent cations (O'Sul-
livan and Perrin, l96h; Wold and Ballou 1951; Mildvan and Cohn, 1966)
and the divalent cation substrate species is thought to be the reactive
substrate species in enzymatic catalysis (Melchior, 1965). It should
be noted that with the concentration of substrates and divalent cations
used (Table 6), the cation-substrate complexes would be present in suf-
ficient concentrations (about 0.1-1 mM) to be considered as a possible
factor in decreasing the rate of inactivation.

It is interesting that fructose-1,6-diphosphate protects and
somewhat destabilizes the control enzyme. It has previously been
shown (see Introduction) to interact with pyruvate kinase from sources
other than muscle; these results show that it binds to rabbit muscle

pyruvate kinase as well.

106

METHODS AND MATERIALS
Rabbit muscle pyruvate kinase was purified and assayed as pre-
viously described (Johnson _£__1,, 1969; Steinmetz and Deal, 1966; Teitz
and Ochoa, 1958). Pyridoxal, pyridoxamine, pyridoxamine-5'-phosphate
and PLP were purchased from Sigma (St. Louis). All other compounds were

reagent grade products from commercial sources as previously described

(Johnson t 1., 1969).

_~

INACTIVATION. Unless otherwise stated, the following procedure was
used: the stock enzyme solution (60 mg per ml) was diluted to 5.h mg
per ml with 0.2 M imidazole, pH 7.5, and dialyzed lO-lh hr at ho versus
150 volumes of the buffer. Following dialysis, the protein was further
diluted to 0.0h2 mg per ml with solutions containing buffer plus the
prOper constituents (see Results). Aliquots (0.1 ug) of the resulting
solutions were then assayed. There was no initial reactivation of the
inactive enzyme by dilution into the assay solution since the assay was
linear for up to l min; however, reactivation was observed in an
extended assay as shown by an increase in the rate of the reaction.

Accordingly, the initial rates were measured.

REDUCTION. The enzyme-PLP complex was reduced by first incubating 1 ml
of enzyme solution (0.h mg per ml) with the apprOpriate PLP concentra-
tion for 15-50 min at 25°, then adding 2 mg of finely divided sodium
borohydride and incubating for 15 min more. The samples were dia-
lyzed for 12-15 hr versus 100 volumes of the buffer at ho. The ratios
of moles of inactivator bound per mole of enzyme were calculated using

molar extinction coefficients of €325 = 10,000 for the é-aminolysine-

107

PLP complex,€ 395 = 9,710 for theE-amino-lysine-pyridoxal complex

-1
(Fischer _3 _a_l_., 1965), andeggo = 0.511 ml mg cm for the enzyme

concentration (Bficher and Pfleiderer, 1955).

CENTRIFUGATION. The procedure used for sucrose density gradient cen-
trifugation followed that of Martin and Ames (1961). The dialyzed
protein was incubated for 15 min with 0.5 mM PLP at 2LI° in 0.2 1_4_
imidazole, pH 7.5; aliquots (h.2 ug protein in 0.1 ml) were layered
on the sucrose density gradients (5-20 86) with the same composition.
Samples were centrifuged for 16 hr at 00 and at h0,000 rpm using a
Spinco Model L Ultracentrifuge with a SW 59 rotor. Fractions of ten
drOps each were collected from the tubes and assayed for pyruvate
kinase activity.

For the reactivation analyses, Tris buffer (pH 7.5, final
concentration, 0.05 M) was added to each fraction before assaying.
The solutions were then incubated for 1 hr at 2&0 and assayed. Con-

trol samples were treated identically, except PLP was omitted.

UV SPECTRA. The UV difference spectra were recorded with a Cary
Model 15 spectrophotometer using double-tandem cells as described by
Kayne and Suelter (1965), and with a Beckmann spectrOphotometer

equipped with a Gilford absorbance indicator attachment.

CHAPTER THREE

STRUCTURAL CHANGES OF RABBIT MUSCLE PYRUVATE KINASE

108

109

ABSTRACT

The sedimentation coefficient for rabbit muscle pyruvate kinase
in the original native state has been determined to be 8:0,w = 9.6 S -
9.8 S by sucrose density centrifugation of a crude extract from frozen
rabbit muscle. A similar experiment with a crude extract from frozen
rabbit liver yielded a value of 82o,w = 7.h S and variable amounts of
a faster sedimenting protein (about 9.5 S) for rabbit liver pyruvate
kinase.

The effect of high pH on rabbit muscle pyruvate kinase was
studied with enzyme preparations from fresh rabbit muscle and from
rabbit muscle frozen 2-5 years. The fresh muscle preparation in 0.05

M Tris or glycine buffer, 0.15 M KCl, 0.001 M EDTA showed: (1) a

single peak in the pH region 7.h-10; (2) at either 50 or 2ho, an

o o
0.18 ,6 ___ 9.6 to 80.18 /o =

initial transition at pH 8.5-9.0 from s
20,w eo,w

9.5 S; (5a) at 5°, a second transition at pH 10.5 and above yielding

a slower sedimenting peak (8.0 S) and an additional peak (h S);

(5b) at 2ho, a second transition at about pH 10.1-10.2, resulting in

2 or more broad peaks in sedimentation velocity experiments. The
enzyme prepared from the frozen muscle under similar conditions showed
different results. (1) At 2ho, the initial transition occurred at
much lower pH (pH 7.9-8.h). (2) The initial transition yielded a more
dissociated or unfolded enzyme, 826?: 96 = 9.h S to 8.7 S, and the
peak broadened much more rapidly during sedimentation velocity experi-
ments. (5) At 2&0, a second transition (loss of well-defined peaks)

occurred at pH 9.8 and above; 0.15 M KCl was required to observe this

effect at pH 9.8, but not at higher pH.

110

The sedimentation coefficient of rabbit muscle pyruvate kinase
prepared from fresh muscle was greatly influenced by salt concentra-

tion. It increased from a value of about 9.5 S in no added salt to a

o
0.18 ,6

maximum value of s
20,w

= 9.6 s in 0.1 E'O-S M KCl (pH 7.5, 5° or
2&0) and 0.05 MfO-6 M (NH4)2504 (pH 8.0, 50). It then decreased with
increasing salt concentration to a value of 3:6,: 56 = 7.65 S in 5.0
M KCl. Similar results were obtained with increasing KCl concentra-
tions at pH 10.0 at 2u°.

Sedimentation equilibrium analysis showed that the decrease in
sedimentation coefficient was due to a dissociation into dimers or
monomers. The results were also consistent with a dissociation-

association rapid equilibrium between monomers (or dimers) and tetramers

and/or octamers.

INTRODUCTION
Several different sedimentation coefficients have been reported
for rabbit muscle pyruvate kinase. Warner (1958) first reported a
value of so , = 10.0h S. This value has been confirmed by a number

of investigators but values as low as 8.5 S have also been reported.

Kimberg and Yielding (1962) reported sedimentation coefficient values

of 820 w = 10.0-11.5 S, determined from sucrose density centrifugation
)
0
experiments and 8:68: ’b = 8.85 S from analytical ultracentrifuge
.9

experiments. Kayne and Suelter (1965) observed a sedimentation

coefficient of 3:0 w = 9.33 s in 0.1 M KCl and 0.001 1~_1 Mn012 and
)

9.22 S in 0.105 M tetramethylammonium chloride. Kayne (1966)

reported an irreversible transition from so = 10-07 to so = 8.55 S
20,w 20,w

upon extended dialysis and also upon extended storage of the enzyme in

111

aqueous buffered solutions at 80 mg/ml at 11°. This transition did not

alter the catalytic activity. A similar time-dependent transition has

also been observed with prolonged storage as the ammonium sulfate sus-

pension of the enzyme (Cottam _t _1., 1969). Kayne and Suelter (1968)

later reported values of 320 w = 8.5-8.9 S depending upon pH and salt
)

species present. Wilson g _a_l. (1967) published sedimentation values

= 9.99 S - 10-17 S in 0.05 M solutions of potassium phosphate,

0
of s
20,W

Tris phosphate, lithium phosphate, or potassium chloride, and 9.55 S

in Tris chloride.
The mol wt of the subunits of rabbit muscle pyruvate kinase has

been determined to be 57,000 (Steinmetz and Deal, 1966). The 10 S

Species (mol wt 257,000; Warner, 1958) can thus be assumed to be a

However, the discrepancy between

tetramer (Steinmetz and Deal, 1966).
the often observed values of about 8.5 S and 10 S, the value generally

assumed to represent native enzyme, is far outside the limits of

experimental error. Furthermore, the explanation cannot be due to

Concentration dependence in those cases not extrapolated to zero
PrOtein concentration. The usual concentration dependence for

g lobular proteins is -0.05 S/mg and almost never greater than -0.1

S/mg,
Two possible explanations for the discrepancy are (l) dissocia-

tion or (2) unfolding. However, neither of these alone is completely

Cons istent with the reported characteristics of the 8.5 S species.
Consider the possibility of dissociation. From the graph of
Bed imentation coefficient versus mol wt for globular proteins

(Figure 2h), 8.5 8 should correspond to a mol wt of about 170,000.

 

112

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115

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Since the "native tetrameric" enzyme has a mol wt of about 250-2h0,000,
this would require an asymmetric dissociation of the tetramer into a
monomer and a trimer; but only one peak has been observed in the sedi-
mentation velocity experiments (Kayne, 1966). However, this could be
consistent if the dissociation involved dimers and tetramers, or
monomers, dimers and tetramers in a rapid equilibrium (Gilbert, 1955).
However, preliminary experiments (Kayne, 1966) show a lack of depen-
dence of the sedimentation coefficient on protein concentration. This
argues against a rapid equilibrium dissociation-association mechanism.
Consider the possibility of unfolding. The 15 86 difference in
sedimentation coefficients is near the maximum that could be expected
for an unfolding; moreover, if the difference were due solely to unfold-
ing, the 8.5 S species would show a greatly decreased sedimentation
Coefficient with increased protein concentration. However, there
appeared to be no significant concentration dependence of the sedi-
mentation coefficient (Kayne, 1966).
Thus, additional studies were necessary to determine the
or iginal in !_i_v_g native state of rabbit muscle pyruvate kinase and to
determine the nature, origin and significance, if any, of the altered,
"non-native" states. With this in mind we undertook a detailed analy-
s is to determine the original native state of the enzyme and study con-
dit ions which could alter it, in an attempt to determine conditions
whereby we could form the 8.5 S from the native state of the enzyme.

The results of this study show that the original native structure has

0
0.16 b

= 9.6 S and that it is reversibly
eo,w

a Sedimentation coefficient, 8

altered by pH, temperature, and salts.

115

RESULTS

Analysis of the Sedimentation Properties of Pyruvate Kinase From Crude

Extracts of Rabbit Skeletal Muscle and Rabbit Liver. Before attempting

to analyze the sedimentation prOperties of purified pyruvate kinase,

the sedimentation coefficient of pyruvate kinase from the crude extract

of frozen rabbit muscle was determined to provide a reference to check

for possible structural alterations during purification. Sucrose den-

sity centrifugation was used for this determination since the pyruvate

kinase could be located by assay measurements, independent of the con-

terminating proteins of the crude extract.

From the data shown in Figure 25, the sedimentation coefficient

of pyruvate kinase from the crude extract of rabbit muscle was deter-

= 9.6-9.8 S using the rabbit muscle aldolase (3:0 w =
- .9

mined to be so
20,W

'7 - 8 S; Deal gt; al., 1965) and rabbit muscle glyceraldehyde-5-phosphate

dehydrogenase (920 w = 7.5 S; Constantinides and Deal, 1969) of the
I

<-‘-1:‘711de extract, as markers.

The sedimentation prOperties of pyruvate kinase from a crude

e3"Et2ract of rabbit liver were also determined (Figure 26). We assumed

the marker enzymes in liver had sedimentation coefficients identical
to their counterparts in rabbit muscle, and determined the sedimenta-
t ion coefficient of liver pyruvate kinase to be approximately 820,W
7 '14- S. This suggests that the rabbit liver pyruvate kinase has a

1°Wer mol wt than rabbit muscle pyruvate kinase; it could be a dimer

1‘18 tead of a tetramer. A small amount of a faster sedimenting species

(about 9.5 S) was also present as indicated by the slight shoulder at

tnbee 7-10 (Figure 26). In other experiments not shown, larger rela-

116

 

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117

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120

tive amounts of the faster moving peak were observed.

Rat liver has been shown to contain two pyruvate kinases of mol
wts 208,000 and 250,000 (Tanaka _£H_l., 196T) (presumably both are
tetramers). Thus, the rabbit liver enzyme may dissociate into a slower
sedimenting species at the low protein concentrations in the extract.

Alternatively, the rat liver pyruvate kinase may differ from the rab-

bit liver enzyme.

Effect of pH of the Sedimentation Coefficient of Rabbit Muscle Pyruvate
Kinase. The series of experiments evaluating the effect of pH utilized
two different enzyme preparations: one prepared from fresh rabbit
muscle and used within one month after preparation, and the other pre-
pared from rabbit muscle which had been frozen for 2-3 years. The use
of the two preparations seemed advisable since the sedimentation
properties of rabbit muscle pyruvate kinase have been shown to under-
go a time dependent transition (Kayne, 1966). This would eliminate
any alteration due to age or storage of the preparation, and a compar-
ison of the results with the two preparations would show any differen-
tzial sensitivity to pH of the fresh and the old enzyme.

Preliminary experiments with the old enzyme preparation showed
that at low pH (pH 3.h, 0.1 g citrate), 0.05 5 NaCl and 0.001 g EDTA,
tic: well-defined peaks could be detected in the schlieren patterns.
licawever, at high pH (0.2 !_glycine) two well-defined peaks with sedi-
‘Dntentation coefficients of about 5 S and 8 S were observed. These
“’£ilues are near those expected for an unfolded monomer (mol wt 57,000)
and a trimer (mol wt 170,000), reSpectively (see Figure 21;). Since

this effect was observed near the pH of 8.5 used in previous studies

121

(Kayne, 1966), and since it raised the possibility of an asymmetric
dissociation of the tetramer into a trimer and monomer, it seemed worthy
of further analysis. In particular, it was of interest to see

whether it occurred at pH values near neutrality.

As a function of pH, the sedimentation coefficient curves of
pyruvate kinase isolated from fresh muscle (Figure 27) showed three
basic characteristics: (1) At 50 and 2h0 a transition from 9.6 S to
9.5 S at about pH 8.5-9.0 was observed. Only one peak could be detected
in the schlieren pattern (Figure 29, a, b). (2) At ho there was a
'marked decrease at pH 10.5 in the sedimentation coefficient, accom-
panied by the formation of an additional peak which sedimented at
about h S (Figure 29, b). (5) At 2h° there was a drastic change in
the sedimentation profile from pH 10.1 to 10.2; there was one well-
defined, sharp peak at pH 10.1 whereas at pH 10.2 there was extensive
dissociation and the pattern was such that no single, well-separated
'peak could be detected (Figure 29, a).

The dissociation shown at pH 10.2 (Figure 27; Figure 29, a) was
reversible (see Methods). A well-defined peak (3 = 9.5 S) was observed
in the schlieren pattern, although a large amount of protein precipi-
tated during the dialysis procedure.

The effect of pH on the sedimentation coefficient of pyruvate

Icinase isolated from the aged frozen muscle was also tested. At h.h
o
0.44 lb =

u"lg/ml and 5° (Figure 28) this enzyme showed a transition from s
1

59 .h S to 8.7 S at pH 9.5-9.8. This decrease in sedimentation coefficient
‘vzas accompanied by a much more rapid broadening of the protein peak than

“9113 observed with the enzyme isolated from fresh rabbit muscle (Figure

122

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Figure 27

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126

Figure 29. SEDIMENTATION VELOCITY PATTERNS OF PYRUVATE KINASE ISOLATED

FROM FRESH RABBIT MUSCLE

The enzyme solutions were prepared and centrifuged as described in
Methods. Sedimentation is from left to right. The sedimentation
coefficient indicated above the peaks are the values at the given
protein concentration, corrected to 200 and water. All samples con-
tained 0.05 M_buffer, 0.001 M EDTA, and, except where indicated,
0.15 M KCl. They were run at 56,100 rpm.
(A) and (B) Eppgg: 1.65 mg/ml, 2&0, glycine, pH 9.6; pictures
taken 12 min (A) and 2% min (B) after attaining speed.
Lower: 1.65 mg/ml, 2&0, glycine, pH 10.2; pictures taken
12 min (A) and 2h min (B) after attaining speed.
(C) yppgg: 1.65 mg/ml, 5°, Tris, pH 7.9; pictures taken 2h
min after attaining speed.
Lower: 1.65 mg/ml, 5°, glycine, pH 10.5; pictures taken
2h min after attaining speed.
(D) Eppggz 1.65 mg/ml, 5°, Tris, pH 7.5, 1.5 M_KC1; pictures
taken 12 min after attaining speed.
nggg: Same as upper except the buffer contained 2.5'!

KCl instead of 1.5 M.

Figure 2"

 

128

50, a). An additional h S peak was observed at pH 10.6.

The effect of pH on the pyruvate kinase from frozen muscle was
also tested at 1.5 mg/ml and 2ho. A transition similar to that shown
in Figure 27 was observed; the sedimentation coefficient decreased
from 8:0,w = 9.6 S to 8:0,w = 9.5 S. However, in contrast, the transi-
tion point was in the region of pH 7.9-8.h, instead of pH 8.5-9.0. A
second transition, the loss of well-defined peaks, occurred at pH 9.8
and above (Figure 50, b); added salt (0.15 M KCl) was required for
this effect at pH 9.8, but not at higher pH. This salt requirement
was not observed at any pH with the enzyme prepared from fresh muscle.

The "aging transition" reported by Kayne (1966) and Cottam gt

31. (1969) was not observed.

Effect of Salt Concentration on the Sedimentation Coefficient of
gyruvate Kinase. The ionic environment of the protein has been shown
to be an important determinant in protein structure (see Jencks, 1969,
for a review). It seemed likely that pyruvate kinase could be sensi-
tive to small changes in ionic strength and that perhaps gross changes
in structure may occur at higher salt concentration. Potassium
chloride was chosen for the analysis because it is a required cofactor
for the catalytic reaction (Boyer _t._l., l9h2) and ammonium sulfate
was chosen because of its use at high concentrations in the purifica-
tion procedure (Teitz and Ochoa, 1958). A further reason for the
choice of the two salts was that rabbit muscle glyceraldehyde-5-
phosphate dehydrogenase is specifically dissociated into dimers by

ammonium sulfate (Constantinides and Deal, 1967) and into monomers by

KCl (Constantinides and Deal, 1968).

129

Figure 30. SEDIMENTATION VELOCITY PATTERNS OF PYRUVATE KINASE ISOLATED

FROM AGED FROZEN RABBIT MUSCLE

The enzyme solutions were prepared and centrifuged as described in
Methods. Sedimentation is from left to right. The sedimentation
coefficients indicated above the peaks are the values at the given
protein concentration, corrected to 200 and water. All samples con-
tained 0.05 M buffer, 0.001 M EDTA and, except where indicated,
0.15 M KCl. They were run at 56,100 rpm.
(A) yppgg; h.h mg/ml, 5°, glycine, pH 9.7; picture taken
ho min after attaining speed.
Lower: h.h mg/ml, 5°, glycine, pH 9.0; pictures taken
ho min after attaining speed.
(B) Eppggz 1.6 mg/ml, 2&0, glycine, pH 9.8; pictures taken
8 min after attaining speed.
£9333: 1.6 mg/ml, 2&0, glycine, pH 9.8, no KCl; pictures

taken 8 min after attaining speed.

 

 

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151

Figure 51 shows the effect of increasing salt concentration on
the sedimentation coefficient of pyruvate kinase isolated from fresh
rabbit muscle. The sedimentation coefficient increased from a value

0.16 $6

of about 9.5 in no added salt to a maximum of 320 w
)

0.1 g-o.5 5 KCl, pH 7.5, 211°, and 0.05 g-O.16 14. (NH4);=SO4. pH 8-0. 5°.

= 9.6 S at

and decreased with increasing salt concentration to a value as low as

o
0.16 /o
eo,w

dimer (mol wt 115,000). The transition at pH 9.5-pH 10.0 (Figure 27)

= 7.65 S at 5.0 M KCl, very close to values expected for a

was observed in 0.15 M KCl, but not in 0.5 M KCl. At pH 10.0 sedimen-
tation coefficient decreased with increasing KCl concentration to a
value of 80.10 ob = 7.5 s in 2.5 M KCl.

20,w -

The decrease in sedimentation coefficient with increasing con-
centrations of KCl and (NH4)pSO4 appeared to be a function of ionic
strength and not of molarity of the solutions (Figure 51). This sug-
gested that the decrease in sedimentation coefficient could be corre-
lated to the activity of water in the solutions. The activity of water

in the K01 solutions was calculated from the osmotic coefficient, 0, of

the solutions (given in Harned and Owens, 1958) by the expression:

‘M
In an"?O = - ¢ T005 m

where: a is the activity of water; M is the molecular weight of water;
O is the osmotic coefficient, and m is the molal concentration of KCl.
However similar data for the (NH4)ESO4 solutions could not be
located. Thus the correlation could not be made.
The results were qualitatively similar with increasing concentra-

tions of both ammonium sulfate and potassium chloride under the condi-

 

152

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155

Figure 51

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tions shown, and also with KCl at ho, not shown, suggesting that the
results were general effects of ionic strength and not specific for

any one set of conditions.

Analysis of Rabbit Muscle Pyruvate Kinase in 5.0 M KCl. The most

 

likely explanations for the decreased sedimentation coefficient in

5.0 M;KCl are a rapid equilibrium dissociation-association system

or an unfolding. With either of these possibilities the sedimentation
coefficient should show a pronounced protein concentration dependence.
In the former case it should increase, while in the latter it should
decrease, with decreasing protein concentration.

To test for a protein concentration dependence, sucrose density
centrifugation was used in the protein concentration range of 5.5 to
0.055 mg/ml with the 5-20 Sb sucrose gradients also containing 5.0‘M
KCl. Contrary to expectation, there was no change in the relative
sedimentation coefficients over the protein concentration range
tested. The results of two extremes of concentrations are shown in
Figure 52. The actual sedimentation coefficient could not be deter-
mined because of the lack of stable markers in 5.0 M KCl.

Since the sucrose may have stabilized the enzyme against disso-
ciation or unfolding, two other methods were used to analyze for these
changes. unequivocal molecular weights can be determined from (1) sedi-
mentation equilibrium experiments, or (2) from the sedimentation and
diffusion coefficients using the Svedberg equation (see Methods).
Accordingly, in simultaneous experiments, the molecular weight of
pyruvate kinase in 5.0 M,K01 was determined by sedimentation equilib-

rium experhments and by a determination of the diffusion coefficient

135

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156

Figure 52

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157
and sedimentation coefficient.

The equilibrium experiments showed (1) rabbit muscle pyruvate
kinase dissociated in 3.0 M KCl, (2) a dissociation-association sys-
tem existed which was dependent upon the concentration of protein in
the cell, (3) possibly there was also random aggregation. Using com-
puter programs (see Methods), it was observed (Figure 33) that at 2.5
mg/ml (top of the cell) the molecular weight was 120,000, whereas at
h.7 mg/ml (bottom of the cell) the molecular weight was 3h0,000. The
weight average molecular weight, over the entire cell, was 232,700,
and the Z-average molecular weight was 600,800.

These results suggested the possibility of a rapid equilibrium
between a dimer (mol wt 115,000) or a monomer (mol wt 57,000) and a
tetramer (mol wt 257,000) or an octamer (mol wt h70,000). A monomer-
octamer equilibrium is the best explanation for the data: (1) The

0.18 96

sedimentation coefficient determined in 3.0 E_KC1 was s?OW
~)

= 7.65 S.
A sedimentation coefficient of 7.65 S should correspond to a molecular
weight of 120,000 (see Figure 2h). However, since the rapid equilib-
rium dissociation-association system yielded a sedimentation coeffi-
cient which was intermediate between the two interacting components,
this necessitated the involvement of the monomer and not the dimer in
the equilibrium. (2) The trend of the molecular weight curve shown in
Figure 3} predicts that at concentrations of about 1 mg/ml or lower,
the average mol wt would be far below 120,000 (the dimer mol wt) indi-
cating that the equilibrium must involve a monomer. (3) Molecular
weights (3h0,000) larger than that of the tetramer were observed, indi-

cating the presence of the octamer. (h) The equilibrium constant cal-

138

 

 

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159

Figure 55

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culated at the two extremes of concentration was most consistent with
a monomer-octamer equilibrium. However, the possibility of an equi-
librium between monomers and tetramers cannot be excluded, due to the

presence of aggregation.

o
The diffusion coefficient was determined to be DZéSS lb = 5.30 x
O )
10 7 cmg/sec. Combining this value with 8:61: lb = 7.65 S, the molecu-
}

lar weight of rabbit muscle pyruvate kinase in 3.0 M KCl was calculated
to be 220,000 using the Svedberg equation (see Methods). This value

was not consistent with the lower molecular weight which should be

 

observed at low protein concentrations (1.6 mg/ml) (Figure 35), but

the effect of rapid equilibrium on the diffusion coefficient has not
been determined. The high molecular weight component may have had a
disproportional effect on the diffusion coefficient, thus leading to

an increased apparent molecular weight.

Correlation of the Change in Sedimentation Coefficient with KCl Con-
centration to Enzymatic Activity. The specific activity of rabbit
muscle pyruvate kinase increased from about 15 with no added KCl in
the assay to a maximum value of 155 in 0.06 M70.l M KCl. It then
decreased to about 50 9b of the maximum activity in 0.6 M KCl
(Figure 3h). Similar activity results have been previously reported
(Melchior, 1965).

These results did not parallel the change in sedimentation
coefficient as a function of KCl concentration (Figure 31). Although
the time of exposure to the salt differed in the two experiments, some
correlations can be made. The activity and sedimentation coefficient

reached a maximum at about the same concentration of KCl (0.1 M) and

 

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then decreased with increasing salt concentration; the decrease in
activity did not correspond to the decrease in sedimentation coeffi-

cient.

Reversal of the Transition. The inactivation and decrease in sedimen-
tation coefficient in high concentrations of KCl was shown to be
reversible. The product of a 12 hour dialysis in 0.15 M KCl, 0.05 M
Tris, pH 7.5 at 2RD had a sedimentation coefficient of 8:0,w = 9.6 8.
Also, the enzyme which should have had little, if any, activity in 2.0

or 5.0 M KCl (Figure 3h) was fully activated by dilution into the

assay mix.

DISCUSSION

This study shows that in its original native state, rabbit

o
0.16 lb 3

muscle pyruvate kinase has a sedimentation coefficient of 820 w
i

9.6 8; this value is also found by a sucrose density analysis of
pyruvate kinase from a crude extract from rabbit muscle. It has
also shown that the native enzyme is reversibly altered by high pH

or high ionic strength. High salt 3.0 M KCl (pH 7.5, 2&0) causes a

o
0.18 ,b
eo,w

decrease in sedimentation coefficient to a value of s = 7.6 S.
Moreover, a molecular weight analysis has shown that this decrease is
due to a dissociation, and the results are most consistent with a
rapid equilibrium between monomers and tetramers and/or octamers.

The range of sedimentation coefficients observed at about pH 10.0-10.5
may also be explained by a rapid equilibrium but these systems have

not yet been analyzed.

We did not observe the irreversible conversion to an 8.5 S

mu

species previously reported (Kayne, 1966). However, we have shown that
rabbit muscle pyruvate kinase appears to exist as a rapid equilibrium
mixture under certain conditions. It is possible that this equilib-
rium may exist under normal conditions of temperature, pH, and ionic
strength and strongly favor the native tetrameric enzyme under the
protein concentrations previously studied. l
The shift for the transition to a lower pH with the aged, '3‘?
frozen enzyme is particularly interesting. It is possible that con-

ditions such as prolonged storage in high salt concentrations or at

 

i

an unusual or unexpected high pH may irreversibly alter the equilib- L;
rium position. This could result from a modification of the enzyme
(i.e., oxidation of sulfhydryl groups) to a form which does not readily
associate.

It is possible that the high salt concentrations used in purifi-
cation may cause dissociation. However, the ammonium sulfate precipi-
tation is done at a higher protein concentration than that of this
study (20 mg/ml) and the effect of this factor on the dissociation is

not known.

Mechanism of Alteration of Structure by Highng and High Salt. Rabbit
muscle pyruvate kinase contains a large number of the hydrophobic
amino acids: alanine, glycine, valine, leucine, and isoleucine (Kayne,
1966; Cottam‘g£.§l., 1969). Hence pyruvate kinase could be expected
to be unstable in a hydrOphobic environment. It has been dissociated
into completely unfolded subunits in the highly hydrophobic environ-
ment of h-8 M urea (Steinmetz and Deal, 1966). But the enzyme is

relatively stable in the lesser hydrophobic environment of ethanol

115

(50 3b), and the reagent is used in the purification procedure (Teitz
and Ochoa, 1958). Recently, Cottam g5 gl. (1969) reported that lower
concentrations of guanidine-HCl (greater than 1 M) than urea (h M) are
required to dissociate the enzyme into completely unfolded subunits,
presumably due to the dual, ionic and hydrophobic, character of the
guanidine-H01 solution. This suggests that hydrOphobic bonding is

not the sole determinate for protein structure, but ionic and hydro- lard
gen bonding may also be important.

It is possible that the dissociation by high concentrations of .-u

 

salt is due to a disruption of key ionic linkages. However, an alter- tj
nate explanation is that the high salt concentrations dissociate the
enzyme indirectly by changing the characteristics of the solvent.
Because urea is able to dissociate rabbit muscle pyruvate kinase
completely into ultimate subunits, it is highly unlikely that ionic
bonds could be a major determinate in subunit bonding. Such has been
found to be the case for hemoglobin (Kirshner and Tanford, 196M;
Kawahara £3 31,, 1965). Two types of binding sites between subunits
were postulated, one ionic and the other hydrophobic. It is likely
that the dissociation by high concentrations of salt is due to a com-
bination of the two alternate explanations.

The dissociation at high pH suggests that a charged group with
a pK of about 10 is important for proper subunit binding. However,
the possibility of base catalyzed cleavage of peptide linkages cannot

be excluded.

The Partial Specific Volume Factor. An exact knowledge of the partial

specific volume (6) of pyruvate kinase is essential for valid sedimen-

lh6

tation and molecular weight determinations. It is assumed in the cal-
culations that the partial specific is constant for all the buffer con-
ditions tested. However, the possibility of preferential interaction
of pyruvate kinase with one member of a multicomponent system, in the
case of the salt species, exists, such that the specific volume of the
enzyme solute complex may be quite different from that of the pure
enzyme.

Previous studies have shown that high salt concentrations do
not affect the partial specific volume of other enzymes. The partial
specific volume of hemoglobin is unchanged in solutions up to 2.65 M
NaCl (Kirshner and Tanford, 196%), and that of human transferin or
bovine plasma albumin does not change in solutions up to 0.2 M KCl
(Hunter, 1967).

Although a slight change in the partial specific volume at
high salt concentrations is possible, it is unlikely that such a
change would be great enough to significantly affect the sedimenta-
tion values. It should be noted that a change in partial specific
volume from 0.7h1 cc/g to 0.795 cc/g would be required to account for

the change in sedimentation coefficient from 0.15 M KCl to 3.0 M KCl.

MATERIAL AND METHODS
Pyruvate kinase was prepared and assayed as previously described

t al., 1969).

(Teitz and Ochoa, 1958; Steinmetz and Deal, 1966; Johnson
The source of the enzyme was skeletal muscle from live rabbits
obtained locally or from frozen muscle (Pel Freez Biologicals, Inc.)

thich had been frozen for 2-3 years (see Test for further details).

1&7

Analysis of the Crude Extracts From Rabbit Muscle and Rabbit Liver.
Frozen rabbit muscle or frozen rabbit liver (5-10 g) was added to an
equal weight of water and homogenized for 15 minutes at 0°. The solu-
tion was then centrifuged at O0 for 15 minutes at 27,000 g. The
supernatant was analyzed by sucrose density gradient centrifugation
(Martin and Ames, 1961). The Spinco Model L ultracentrifuge was used
and the SW 59 rotor was run at 00 for 1h hours at h0,000 rpm. Aldo-
lase and lactate dehydrogenase, which served as markers, were assayed
according to the procedures of Richards and Rutter (1961) and Chasin

(1967), respectively.

Preparation of the Enzyme for Ultracentrifugal Analysis. The stock
enzyme solutions (70-90 mg/ml) were diluted to 1.65 mg/ml or h.h mg/ml
with a solvent containing 0.05 M Tris (HCl) or glycine (K) buffer,
0.001 M EDTA and the indicated salt concentration. They were then
dialyzed for ll-15 hours at 50 or 2h° against 125 volumes of the
buffer. The ammonium sulfate solutions were adjusted to proper pH
with H2804, and the other solutions were adjusted with HCl or KOH.
Except for the ammonium sulfate solutions, the pH of the samples was
adjusted to the prOper pH at 2&0, and the actual pH at the lower tem-
peratures was calculated using the correction factor, -0.05 pH units
per degree (Long, 1961). This correction factor was also verified
experimentally. Thus, for all except the ammonium sulfate samples,
the pH values listed are the actual values at the temperatures at
which the experiments were performed. Due to the presence of varying
concentrations of ammonium ions in the ammonium sulfate solutions,

the dependence of pH with temperature was not determined and the pH

1h8

. 0
reported is that measured at 2h .

Ultracentrifugal Analysis. All experiments were performed in a Spinco
Model E ultracentrifuge equipped with phase-plate schlieren optics.
The exact temperatures were obtained with a calibrated rotor and tem-
perature indicator and control (RTIC) unit. Photographic plates were
measured with a Bausch and Lomb microcomparator. Calculations were
done manually or on a Control Data Corporation 5600 digital computer,

using computer programs deve10ped in this laboratory.

Sedimentation Velocity Analysis. Sedimentation velocity experiments
were done with 12 mm signle sector cells, at 56,100 rpm in an An-D
rotor. The sample volumes were approximately 0.55 ml. Sedimentation

coefficients were calculated from the expression:

1 r (t)
S = 2 1n -2-?——7
(t to)w rp to
where S is the sedimentation coefficient in Svedberg units (10-13
seconds = S), rp is the peak maximum in the refractive index gradient

curve of the picture taken at time to (first picture taken), or at

time t (later pictures), and w is the rotor velocity in radians/sec.

Sedimentation Equilibrium Analysis. Standard double sector cells were
used for the sedimentation equilibrium experiments. Equilibrium was
reached after centrifuging for 2h hours at 699k rpm and 2&0 using an
An-D rotor. Computer programs developed in our laboratory were used
to calculate weight-average and Z-average molecular weights, averaged

over the whole cell, and also the protein concentration and "point"

 

1A9

weight-average molecular weights at various radial positions in the

cell. The "point" mol wt (M) was calculated using the expression:

M = A'RT . (dc/dr)i
(l-vQ )w2 rc
where T is the absolute temperature, R is the gas constant of 8.51% x
107 ergs/mole/degree, G is the partial specific volume of the protein,
Q is the density of the solution, w is the angular velocity of the
rotor in radians/sec, and c and dc/dr are the concentration and con-

centration gradient at the point, r, in the cell.

Diffusion Coefficient Analysis. The diffusion coefficient, D, was
determined with a 12 mm double sector synthetic boundary cell at
h,059 rpm using an An-D rotor. Values were calculated manually from
an earlier equation (Schachman, 1957), modified to allow use of mea-

sured data without correction for the magnification factor, F:

__ m
D- #1760192

where m is the slope of a line from a graph which plots area2/height2

versus time (minutes).

Molecular Weight Determination Using the Svedbergtgguation. The
molecular weight is calculated from the sedimentation and diffusion
coefficients using the following equation (Svedberg and Pedersen, 19h0):

8 RT

M: 0(1-69)

where the terms are the same as those defined above.

 

150

Buffer Correction Factors. (See Appendix II for a detailed analysis.)
The Viscosities of the solutions were determined in constant temperature
water baths by means of a Cannon-Ubbeholde capillary dilution viscometer.
Densities of the solvents were determined by use of a pyncnometer. The
value of 0.7hl cc/g at 200 (Kayne, 1966) was used for the partial speci-
fic volume; a change of 0.005 units per degree (Hunter, 1966) was used
to correct for temperatures other than 20°. The change in viscosity

due to temperature was determined from published tables (Handbook of

Chemistry and Physics, 1965).

SUMMARY AND CONCLUSIONS

(1) The dissociation of tetrameric rabbit muscle pyruvate kinase
into unfolded subunits has been shown to be reversible. This study
confirms and greatly extends the preliminary studies reported by Kayne
(1966). The reversal is accomplished by a 100-fold dilution of the
dissociated enzyme solution into a renaturation mixture. Optimal con-
ditions for the reversal (up to 70 96 recovery of activity) are
(a) pH 8.0; (b) protein concentration, 0.0M mg/ml; (c) ionic strength,
0.5; (d) reducing agent, 0.06 M B-mercaptoethanol; (e) temperature-0o
dilution, followed by 6 hr at 16°. Insulin decreases the extent of
the reversal, and phosphate is required for reversal at 56°. None of
the other metabolites tested influenced the reversal.

The reasons for the lack of higher than 70 96 recovery of activ-
ity are not known. Perhaps insulin may aid the folding under sets of
conditions or combinations of metabolites different from those tested.

(2) Rabbit muscle pyruvate kinase is reversibly inactivated by
a time dependent (half-time of 5 min) formation of a Schiff base with
the biological cofactor, PLP. Reduction of the inactivated complexes
shows that there are two types of binding, (1) a specific inactivating
binding, involving 2-h moles of PLP, (2) a non-specific noninactivat-
ing binding.

(5) Glutathione and FDP were found to interact with the enzyme

btlt these phenomena were not studied in detail.

151

 

kinas

mined

vari'

samp

crud

has

P?“

at

‘4; )

mo

152

(h) The sedimentation coefficient of rabbit muscle pyruvate
kinase in its original (and presumably ig_yigg) state has been deter-
mined to be s26}: 96 = 9.6 S since this is the value found under a
variety of conditions with a number of different preparations and
samples. Results from sucrose density centrifugation analysis of
crude rabbit liver extract showed that rabbit liver pyruvate kinase
has an 8:0,w = 7.h 8; this is lower than the value expected for

pyruvate kinase from rat liver (Tanaka g£_§l., 1967).

(5) The effect of high pH on the sedimentation coefficient of

 

pyruvate kinase has been evaluated from enzyme isolated from fresh
rabbit muscle and from muscle which has been frozen for 2-5 years.
Both samples showed a change in sedimentation value from 9.6 S to
9.5 S at about pH 8.5-9.0 with an additional transition to about 8.5 S
at pH 10.5 and at 5°. With the aged frozen enzyme the transition to
9.5 S could be observed as low as pH 7.9 at 2ho. The aged frozen
enzyme was unstable at pH 9.8 and above at 2ho;the loss of peaks
was observed in the presence of salt but not if there was no salt
present.

(6) The sedimentation coefficient increased from 9.5 S with no

0.16 96

added salt to a maximum of sgo’w = 9.6 S at 0.1-0.5 M KCl. It then

decreased, without gradations, at higher salt concentrations, reaching

0.16 96

20 w a 7'65 S in 5.0 M KCl. This transition was rever-
.9

a value of s
sible by dilution or by dialysis.
(7) Sedimentation equilibrium analysis shows that the decrease

ill sedimentation coefficient is due to a dissociation into dimers or

fluinomers. The results of this study are also fairly consistent with a

155

dissociation-association rapid equilibrium between monomers (or dimers)
and tetramers and/or octamers.

(8) The conditions necessary for the observed production of a
more slowly sedimenting species (8.5 S) (Kayne, 1966) at neutral pH in
aqueous solutions has not been defined. A possible explanation for
this irreversible transition is an alteration, such as oxidation of a
critical sulfhydryl group, during prolonged storage of the enzyme at
high salt concentrations or high pH.

(9) The results of the studies on the native structure of rabbit

muscle pyruvate kinase are consistent with the following model:

 

 

17 S
pH salt :0
T < ’ *—> [1‘ D é.__> by
10.07 S 10.07 S 7.5 S 5.6 S

[we <—————> .. <—>d
(8-5 8)

where: T represents the native tetrameric native enzyme (mol wt
257,000, 10.07 S); D represents the dimer (mol wt 115,000, 7.5 S);
M represents the monomer (mol wt 57,000, 5.6 S); 0 represents the
octamer (mol wt h70,000, 17 S); and T* represents the irreversibly
altered native enzyme which enters into the equilibrium leading to

‘the 8.5 S sedimentation coefficient.

APPENDIX I

Derivation of Rate Equations Used to Study the Rate of

Inactivation of Pyruvate Kinase by Pyridoxa1g5'-Phosphate

The rate of inactivation of pyruvate kinase by pyridoxal 5'-

phosphate can be expressed by the general equation:

(1) - ff} = km + x) (PLP)“<A)"

where: A is the active enzyme concentration; PLP is the concentration
of pyridoxal 5'-phosphate; n is the order of the reaction with respect
to PLP concentration; x is the order of the reaction with respect to
active enzyme concentration; and k is the (n + x)th order rate constant
for the inactivation.

If we assume x = 1; and (PLP) is constant,* expression (1)

reduces to:

(2) --%:—=k' (A)

this can be rearranged to:

A v
(3) --a—=kdc

where k', the "pseudo first-order rate constant," is equal to:

 

-7

*Since the concentration of_enzyme is 1.75 x 10 moles/liter
and the concentration of PLP is 10 4 moles/liter, this is a valid
assumption.

15h

 

 

L__.__.

 

Upon int

Raw 3 p

of this

Since 1

The re
of a I
VETSU

react

If n

haVe

155

(h) k' = k( ) (PLP)n

n + 1
Upon integration, expression (5) yields:
(5) 1n A/AO = -k't

A
Now a plot of 1n /Ao* versus time yields a straight line; the slope f

of this line is equal to -k'.

Jun-.1. -—-——-

Since k' = k( (PLP)n

n + 1)

 

(6) log k' = log k( + nlog (PLP) ;

n + l)

The rate of the reaction with respect to PLP,n, is given by the sIOpe
of a plot of the log k}, determined at various concentrations of PLP,
versus the respective concentration of PLP. The rate constant for the

reaction k( is the intercept of the k' coordinate.

n + 1)’

If n = l, the plot of k' versus the respective PLP concentration should

have a 810pe equal to the second-order rate constant for the reaction.

 

*This is equivalent to the In of per cent activity remaining.

APPENDIX II

Determination of the Correction Factors Necessary for Correcting

the Observed Sedimentation Coefficients to Standard Conditions I
mg

The sedimentation coefficients observed under various conditions 7

of solvent composition and temperature were corrected to the standard a

 

conditions of 200 and water. This appendix explains in detail the '
determinations and calculations used for these corrections. One

result was surprising and of special interest. In the viscosity deter-

minations the flow times for the KCl solutions were shorter than those

for the reference solvent, water, which is most unusual. This confirms

the results from the International Critical Tables (1929), which show

this effect indirectly by listing relative Viscosities of less than

unity. Another important result was that the Viscosities of the KCl

solvents relative to water were not independent of temperature.

The Correction Expression, The complete expression for the correction
of the sedimentation coefficient to 200 and water is given by the fol-
lowing equation:

= 2010920171
E...) Ali-n.5,? <w>

where: s2o w is the sedimentation coefficient corrected to the stan-
)

dard conditions; st b is the observed sedimentation coefficient mea-
)

156

157

sured at the temperature, t, and in the solvent buffer, of the experi-
Y\t,b . .
ment; ( /V\oo w) 18 the visc051ty of the buffer at temperature, t,
' )
relative to that of water at 200, Q 20 w is the density of water at 20°;
)

Q is the density of solvent at temperature, t; G is the partial
t,b 20,w

specific volume of the protein in water at 20°; and G is the partial

t,b
specific volume of the protein in the buffer at temperature, t.

Mb,

The term, is composed of two terms:

t,w

Eqn(2)1\_t£_ x 112.12
Y\2o,w Y\t,w

The first term is the temperature contribution to the viscosity
of water. The values are found in the Handbook of Chemistry and
Physics (1965). The second term is the viscosity of the buffer rela-
tive to that of water. It is generally assumed to be independent of
temperature, but the results of this study have shown that this is
not true for high concentrations of KCl. This term is determined by

the following expression showing the temperature dependence.

“t,b

Eqn (3) New = (Qb/Qw) (Tm/Tm)

where (Qb/ Qw) is the density of the buffer solution relative to that

of water and (Tt b/Tt w) is the flow time of the buffer solution
) 1

through the viscometer relative to water at temperature, t.

The density, Q (Eqn (1)) of the solvent at temperature, t,

t,b
is approximately equal to the relative density, Q t/Qw’ since the
value of Q’w is very close to unity, ranging from 0.99997 g/cc at 60

to 0.99825 g/cc at 200 (Handbook of Chemistry and Physics, 1965). Thus

158

a determination of the relative density factor eb/ 9w is sufficient to

determine Q t b'
)

. are found i th
The correction factors (nt,w/ 1120””) and 920,“, n e

Handbook of Chemistry and Physics (1965). The partial specific volume

has been discussed in Chapter 5 and will not be further mentioned. The

 

 

remainder of this Appendix will discuss an evaluation of the y‘t,b/)1t,w .1
and e b/ PW factors. “
1
Necessity for Additional Determinations. The relative viscosity and .;
density terms for several ammonium sulfate and potassium chloride solu- .J

tions are given in the International Critical Tables (1929). However,
the tables are not complete for the salt concentrations and temperatures
used in the centrifuge experiments of Chapter 5. Moreover they do not
include the contributions of the remaining buffer components (0.05 M
buffer and 0.001 M_EDTA) to the density and viscosity.

For these reasons we undertook a detailed analysis to determine
the relative Viscosities and relative densities of the buffer solutions

used in Chapter 5.

Results. The Viscosities of the solutions were determined in constant
temperature water baths by means of a Cannon-Ubbelodhe capillary dilu-
tion viscometer. Densities of the solvents were determined by use of
a pyncnometer.

The relative densities of the solutions are shown in Figure 55,
and the relative flow times through the viscometer in Figure 56. From
these values, the relative Viscosities were calculated (Figure 57).

The relative Viscosities of a few representative solutions with

159

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monHDAOm eu<m mes mo mmHeHmzma m>He<umm .mm muswsm

160

Figure 55

C350: vommvzzv
2 «3 no go o
4

A .
>:¢<._O<< .8.
0d mN QN m.—

 

 

 

 

. . u‘ ». .... k u 9 -.rOL 5.. . ..4.*o a v . v . 1 k

a“ ....I. o ..L. . .OI'f. 1 ..',1‘n.l.—' %.o-lll..1.

s .1? 0.. v6 4, THHM 90?! n M ‘F; s x Q” .6. u o . -.H51I4Olm 1.“.
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.4: L 0 .‘IY.'|I. O ‘J o ... 4 o O O as v . 6 . r... . L... O 4

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I ’v‘...‘. L --

o ‘ {Ir . . .

 

r.'¢.v.u.. o
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oil--- .11
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161

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mmHmZOUmH> mmH EODOMSH mZOHHDAOm HA<m mmH m0 MZHH 30am m>HH<Amm .om munwwm

162

Figure 56

32.302 womuxvzzv
8 m3 no go

a a
>2¢<a0<< .3.
0d nN ON 3 Q— md

.
a “+044 ‘41:
.11 '9' <.. .
to. .0... $6.. ..
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1!... - o 1..
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.6... '..v”. .0.

..- , .n

 

mgr—Dag ....(m “.0 32:. 30.: w>=<aw¢

a:

I

165

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”conmoumxo

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Figure 57

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2 m3 ....o 3o

4 . _
>:¢<._0<< .3.

a s ' cor.
V; llot .13".
0.0:. 93191..

c.. c ¢.6 t...‘ o
-

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....

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'

3:22.38 ...—(m “_0 mw_.—.m0Um.> w>=.<._w¢

 

165

glycine buffer replacing the Tris were determined and were found not to
significantly differ from these values. The relative densities increased
with increasing salt concentration of both KCl and (NH4)2SO4. However
the flow time through the viscometer showed different results. The solu-
tions with increasing concentrations of KCl actually flowed through the
viscometer faster than water, and the effect was even greater at 6.100
than 25.650. ‘This confirms the results reported in the International
Critical Tables (1929), but it is interesting that this is contrary to
what has been reported for NaCl which flowed through the viscometer
slower than water (Svedberg and Pedersen, l9h0). The results also

show that the relative viscosity of the K01 solutions are not indepen-
dent of temperature; the relative viscosity of the KCl solutions at
6.100 are significantly lower than those at 25.650. This is contrary

to what is normally assumed for viscosity corrections.

 

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Anderson, B. M., Anderson, C. D., and Churchich, J. E. (1966),
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Anson, M. L. (1951), _J_. Gen. Physiol. g3, 599. a

.-"I .1".‘E”h

Bailey, E., and Walker, P. R. (1969), Biochem. l. 111, 559.

”'I"

Bellau, B., and Burba, J. (1960), ,1. Amer. Chem. Soc. §_2_, 5721.

1"."
..-
-.

Benesch, R. E., Benesch, R., and Yu, C. I. (1969), Federation Proc.
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Blakely, R. L. (1955), Biochem. _J. _61, 515.
Boyer, P. D. (1962), Enzmes _6, 95.
Boyer, P. D. (1969), Biochem. Bighfi. Res. Commun. 1+, 702.

Boyer, P. D., Lardy, H. A., and Phillips, P. H. (19112), _J. Biol. Chem.
116, 675.

Braunstein, A. E., and Skemyakin, M. M. (1955), Biokhimiya _l_8, 595.

Bucher, T., and Pfleiderer, G. (1955), in S. P. Colowick and N. 0.
Kaplan (Editors), Methods E Enzmology, Vol. I, Academic
Press, Inc., New York, N. Y.

Chasin, M. (1957), gh,p, Thesis, Michigan State University.

Constantinides, s. M., and Deal, w. c., Jr. (1967), lihch Natl. ACS
Meeting, Chicago, Abstract 5-198.

 

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