EFFECTS OF TEMPERATURE. SUBSTRATES.
CATIONS, AND FRUCTOSE 1,6-DIPHOSPHATE
ON THE CONFORMATIONS. SUBUNIT
STRUCTURE, AND STABILITY OF
YEAST PYRUVATE KINASE

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
RONALD THOMAS KUCZENSKI

1970

 

 

 
  
 

IIIIIIIIIIIIIIIIIIIIIIIIIIIIWIII

3 1293 00676 7846

  

LI B R A RY
Michigan State
- University I

 
  

This is to certify that the

thesis entitled

EFFECTS OF TEMPERATURE, SUBSTRATES, CATIONS,
AND FRUCTOSE 1,6—DIPHOSPHATE ON THE
CONFORMATIONS, SUBUNIT STRUCTURE, AND STABILITY
OF YEAST PYRUVATE KINASE
presented by

Ronald Thomas Kuczenski

has been accepted towards fulfillment
of the requirements for

Pb .D.dbgree inJiochemis try

Wag."

Major professor

Date July 14‘ 1979_

0-169

 

 

 

  
 
 
      
   

 
 

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ABSTRACT

EFFECTS OF TEMPERATURE, SUBSTRATES, CATIONS, AND
FRUCTOSE 1,6-DIPHOSPHATE ON THE CONFORMATIONS,
SUBUNIT STRUCTURE, AND STABILITY OF YEAST PYRUVATE KINASE

by Ronald Thomas Kuczenski

This thesis describes the native and subunit molecu-
lar weights of yeast pyruvate kinase (PK), and the struc-
tural and conformational properties of the enzyme under
the influence of temperature, substrates, and effectors.

Yeast PK has a molecular weight of 162,000 to
168,000 as calculated with the Svedberg equation under two
sets of solvent conditions. The enzyme sediments as a
single symmetrical peak with an Ego,w of 8.85 S and a
‘230,w of n.84 x 10‘7 cm2 sec-1 in 0.1 M tetramethylammonium
cacodylate buffer, pH 6.2, containing 0.1 M KCl, 2.6 x 10‘2 M
M3012. and 10'3 M fructose 1,6-diph08phate (FDP). In 0.1 M
TriS'HCl, pH 7.5. the values obtained are 8.3“ S for Ego,w
and #.52 x 10"7 cm2 sec’1 for 220,w' Using the high Speed
equilibrium technique at low protein concentrations, a
molecular weight near 165,000 was estimated for the enzyme
in 0.1 M TrisoHCl, pH 7.5, containing 0.23 M KCl, 2.5 x 10"2 M
MgClZ, 2 x 10"3 M FIT, and 10"2 M phoSphoenolpyruvate (PEP).

The enzyme is a tetramer, each polypeptide chain hav-
ing a molecular weight of 02,000 to “5,000. Complete disso-
ciation was obtained in 6 M guanidine hydrochloride-0.15 M

2—mercaptoethanol. Utilizing sedimentation equilibrium under

Ronald Thomas Kuczenski
these solvent conditions, similar values for M3 and M; were
obtained, indicating the subunits have approximately equal
molecular weights. Dissociation was also obtained by
extensive treatment of the enzyme with maleic anhydride,
resulting in a symmetrically sedimenting peak with Mats/Q)
of h2,200, excluding bound maleyl groups.

The tryptophyl fluorescence of yeast PK is quenched
by the addition separately or together of the activating
cations K+ and Mg2+. The quenching is minimal, however,
even in the presence of the substrate, PEP, when compared to
the quenching observed in the presence of the activator, FDP,
either in the presence or absence of the cations or PEP.
Titration of the enzyme with FDP in the presence of Mg2+
monitored by the fluorescence change reveals a marked depen-
dence of the FDP binding constant on the nature of the effec-
tors present. Addition of 0.23 M K+ increases the apparent
KD for FDP from 0.48 mM to 3.1 mM. (CH3)4N+ has a similar
though smaller effect. On the other hand, the addition of
PEP markedly reduces the apparent KD for FDP to 0.069 mM.

K+ is required to obtain the reduced KD, whereas (CH3)4N+
will not function. Adenosine 5'-diph03phate also promotes a
decrease in the apparent KD for FDP. but no monovalent cation
requirement is observed. Changing the temperature from 30 to

00

in the presence or absence of K+ or PEP decreases the
apparent KD for FDP by an order of magnitude.
Yeast PK has also been shown to be susceptible to

inactivation at low temperatures. Addition of micromolar

Ronald Thomas Kuczenski
amounts of the allosteric activator, FDP, markedly enhances,
by as much as 1000-fold, the rate of loss in activity both
at 0° and at 23°. Addition of Mg2+ prevents the inactiva—
tion. At both temperatures a biphasic inactivation results,
with the rate of both steps increasing as the protein con-
centration is decreased.

At 0° the inactivation is accompanied by a decrease
in the sedimentation coefficient of the enzyme from 8.6 S to
3 S followed by a slower decrease to 1.7 S. Both the extent
and the rate of the first step are dependent on protein and
FDP concentrations, consistent with the establishment of an
inactivation equilibrium between a tetrameric and a dimeric
form of the enzyme, involving the binding of at least two
moles of FDP with an apparent geometric average dissociation
constant of 63 uM.

On the other hand, at 23°, only the rate of the first
step of the inactivation is dependent on protein and FDP
concentrations. The extent of inactivation proceeds to loss
of half of the activity of native enzyme independently of
protein and FDP concentrations. This step is accompanied by
the formation of an apparent dimer with._s_20,W = 4.2 S, and
is followed by dissociation to inactive subunits.

Both steps of the inactivation at 23° are dependent
on.FDP concentration, the binding of which is inhibited by
high ionic strength. In the absence of added ionic strength,
FDP binds to the enzyme with an apparent geometric average

dissociation constant of 0.66 mM. This value is increased

Ronald Thomas Kuczenski
to 5 mM at 0.934 u. While the rate of the first step of the
inactivation is independent of ionic strength at saturating
FDP, the rate of the second step is markedly inhibited.

The overall mechanism of inactivation involves the
binding of at least two moles of FDP. followed by a two-step
dissociation of the native tetramer to inactive subunits.
The ionic strength effects, the temperature effects, and
the different time-course of inactivation at the two tem-
peratures, however, suggest heterologous subunit interac-
tions leading to different dimers at O0 and at 23°. These
data are consistent with the inclusion of a large number of
hydrophobic bonds between the subunits of the 23° dimer,
and in the association of the 0° dimers in the native
tetramer.

The analogous effects of temperature and ionic
strength on the binding of FDP to yeast PK as observed
through measurement of fluorescence changes and through the
inactivation phenomenon suggest that similar structural
transitions of the enzyme accompany both the fluorescence
change and the inactivation and involve eXposure of pre-
dominantly hydrophobic residues of the polypeptide chains.
These effects are discussed with reSpect to the mechanism

of kinetic activation of yeast pyruvate kinase by FDP.

EFFECTS OF TEMPERATURE, SUBSTRATES, CATIONS, AND
FRUCTOSE 1,6-DIPHOSPHATE ON THE CONFORMATIONS,
SUBUNIT STRUCTURE, AND STABILITY OF YEAST PYRUVATE KINASE

By

Ronald Thomas Kuczenski

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Biochemistry

1970

To Sharon and Tracy

11

ACKNOWLEDGMENTS

I thank Dr. Clarence H. Suelter for the freedom of
development which he allowed me, as well as for his encour-
agement and guidance throughout the four years required to
complete this thesis. I am eSpecially grateful for the
amount of time he Spent in both scientific and other-world
discussions.

I also eXpress my appreciation to Dr. George M.
Stancel, Dr. James Hunsley, and Dr. Karl L. Smiley, Jr. for
helpful discussions.

Financial assistance in the form of a predoctoral
fellowship from the National Institutes of Health is

acknowledged.

111

TABLE OF CONTENTS

INTROch ION C O O O O O O O O O O O O O O C O O O

LITERATIJHE SIJRVEY O O O O O O O O O O O O I O O 0

Cold Lability . . . . . . . . . .
Metabolite Induced Inactivation . . . . . . .

METHODS
I.

II.
III.

RESUETS
I.
II.

III.

AND HATER IA 15 O O O O O O O O I O O O O O

Enzymes . . . . . . . . . . . . . . . . .
Chemicals 0 O O O O O O O 0 O O O
EIperimental Methods . . . . . . . . . .
1. Assay of activity and determination
of enzyme concentration . . . . . . .
2. Ultracentrifugal analyses . . . . . .
3. Fluorescence analyses . . . . . . . .

Temperature Stability of Yeast Pyruvate
Kinase . . .
Molecular Weight of Yeast Pyruvate Kinase
1. Native enzyme . . . . . . . . . . .
i. s/D molecular weights . . . .
11. High Speed equilibrium technique
2. Subunits . . . .
1. Enzyme in guanidine hydrochloride
ii. Maleylated enzyme . . . . . . . .
3. Effects of cations, substrates, and
fructose 1 ,6—diph08phate on the sedi-
mentation coefficient of yeast pyru-
vate kinase . . . . . . . .
Effects of Substrates, Cations, and
Fructose 1 ,6-DiphOSphate on the Fluores—
cence of Yeast Pyruvate Kinase . . . . .
1. Qualitative effects . . . . . .
2. Titration of yeast pyruvate kinase
with fructose 1,6-diph03phate by
measurement of fluorescence change .
Fructose 1,6-Diph03phate Induced Inacti-
vation of Yeast Pyruvate Kinase . . . . .
1. Inactivation at 0°. . . . . . . . . .

iv

Page

11
16
16
17
17
17
18
20

21

”3

50
50
54

62
62

Page

1. Dependence of the inactivation on
fructose 1 ,6-diphOSphate concen-
tration . . . . 62
11. Treatment of data from. Figure 16 . 65
111. Effect of protein concentration
on the inactivation . . . . . 77
iv. Sedimentation coefficient of inac-
tivated enzyme . . . . . . . . . . 77
2. Inactivation at 23°. . . . . . . . 8h
1. Dependence of the inactivation on
fructose 1 ,6-diph03phate concen-
tration . . . . 84
ii. Treatment of data from Figure 23 . 84
111. Effect of protein concentration

on the inactivation . . . . . 92
iv. Effect of ionic strength on the

inactivation . . . . . 92
v. Sedimentation coefficient of inac-

tivated enzyme . . . 104

vi. Attempted reversal of the inacti-
vated enzyme . . . . . . . . . . . 105

DISCUSSION . . . . C O O O C O O O O O O O O C O O C 1 07
LIST OF REFmENCES . C C O I O O O O O O O O O O O O 131

Figure
1.

10.

11.

12.

LIST OF FIGURES

Effect of temperature on the stability of
yeast pyruvate kinase . . . . . . . . . . . .

Effect of preincubation at 23° on the sta-
bility of yeast pyruvate kinase at 0° . . . .

Sedimentation coefficient of yeast pyruvate
kinase as a function of protein concentra-
tion at pH 6.2 in the presence of KCl,

M8012, and Fm at 200 o o o o o o o o o o o o

Diffusion coefficient of yeast pyruvate
kinase as a function of protein concentra-
tion under the conditions of Figure 3 . . . .

Sedimentation coefficient of yeast pyruvate
kinase as a function of protein concentra-
tion in 0.1 M Tris-H01, pH 7.5 . . . . . . .

Diffusion coefficient of yeast pyruvate
kinase as a function of protein concentra-
tion under the conditions of Figure 5 . . . .

Number-average molecular weight of yeast
pyruvate kinase as a function of fringe
concentration . . . . . . . . . . . . . . . .

Determination of the homogeneity of yeast
pyruvate kinase at 1.75 ms/ml in 6 M GuHCl .

Extrapolation of the apparent weight average
and apparent g—average molecular weights of
yeast pyruvate kinase to zero protein in

6 M GuHCl O O O O O O O O O O O O O O O O O O

Sedimentation coefficient of yeast pyruvate
kinase after maleylation . . . . . . . . . .

Diffusion coefficient of yeast pyruvate
kinase after maleylation . . . . . . . . . .

Fluorescence emission Spectrum of yeast
pyruvate kinase at 23° . . . . . . . . . . .

vi

Page

22

24

28

30

32

34

36

39

41

44

46

52

Figure

13.

14a

15.
16.
17h

1.8.

1.9.

23

2L.

335

26

Plots of the binding of FDP to yeast pyruvate
kinase as monitored by fluorescence changes
(Kcl effeCtS) O O I O O O O O O O O O O O l C

Plots of the binding of FDP to yeast pyruvate
kinase as monitored by fluorescence changes
[(CH3)4NC1 effects] . . . . . . . . . . . . .

Effect of temperature on the FDP-enhanced
inactivation of yeast pyruvate kinase . . . .

Effect of FDP concentration on the stability
of yeast pyruvate kinase at 0° . . . . . . .

Example of the method of treatment of data
from Figllre 16 o e o o o o o o o o o o o o 0

Determination of the apparent KD at 0° of
FDP for yeast pyruvate kinase from inacti-
vat ion data 0 O O O O O O O O O O O O O O O 0

Comparison of eXperimental data with theo-
retical determinations of the zero time
intercept and calculated rate constants at

00. O O C O O O O O I O O O O O O C O O O O 0

Determination of the kinetic interaction
constant between.FDP and yeast pyruvate
kinase at 00 O O O O O O O O O O O O O O O I

Effect of protein concentration on the FDP-
enhanced inactivation of yeast pyruvate
kimse at 0° 0 O O O O O O O O 0 O O O O 0

Determination of the order with reSpect to
protein of the FDP-enhanced inactivation of
yeast pyruvate kinase at 0° . . . . . . . . .

Effect of FDP concentration on the stabil-
ity of yeast pyruvate kinase at 23° . . . . .

Determination of the apparent KD at 23° of
FIT for yeast pyruvate kinase from the fast—
step inactivation data . . . . . . . . . . .

Plot of the slow step ingctivation constants
at 230 as 1/33 E. l/FIP .1 o o o e o o o o 0

Effect of protein concentration on the FDP-
enhanced inactivation of yeast pyruvate
kinaseat230.......

vii

Page

55

6O

63

67

69

72

75

78

80

82

85

88

9O

93

Figure
27.

28%

29,

30.

.31.

Page

Determination of the order with reSpect to
protein of the FDP-enhanced inactivation of
yeast pyruvate kinase at 23° . . . . . . . . . 95

Stabilizing effect of high concentrations
of FDP on yeast pyruvate kinase at 23° . . . . 97

Effect of monovalent cations on the FDP-
enhanced inactivation of yeast pyruvate
kinase at 230. O 0 O O O O O O O O O O O O O O 100

FDP-enhanced inactivation of yeast pyruvate
kinase at 23° at constant ionic strength . . . 102

Preposed mechanism of inactivation of yeast
pfluvate kinase 0 O O O O O O I O O O O O O O 119

viii

Tablus
I.

II.

III.

'VI.

III]:.

LIST OF TABLES

Sedimentation and diffusion coefficients of
native and maleylated yeast pyruvate kinase
as a function of protein concentration . . . 48

Sedimentation coefficient of yeast pyruvate
kinase as a function of cations, substrates
and FWD 0 O O O O O O 0 O O O O O O l O O 0 1+9

Relative fluorescence of yeast pyruvate
kinase as a function of cations, PEP, and
Fm O O O O O I O O O O O O O O O O O O O I 51

Apparent dissociation constants of yeast
pyruvate kinase and FDP obtained by titra-
tion of fluorescence changes . . . . . . . . 57

Requirement for FDP for enhanced inactiva—
tion of yeast pyruvate kinase . . . . . . . 66

Rates of inactivation of yeast pyruvate
kinase . . . . . . . . . . . . . . . . . . . 87

Effect of temperature on the apparent KD of

FDP for yeast pyruvate kinase from fluores-
cence changes and inactivation data . . . . 114

ix

ADP
ATP
CHA

220,w

o
Ebo,w
DHAP

FDP

G-BAP

GTP

GuHCl

KD

(KI) g.ave.
34..

£4.13

flgtgfly)

LIST OF ABBREVIATIONS AND SYMBOLS

Adenosine 5'-diph08phate
Adenosine 5'-triph05phate
cyclohexylammonium cation

diffusion coefficient corrected to 20° and
water

diffusion coefficient corrected to 20° and
water and extrapolated to zero protein con-
centration

dihydroxyacetone phOSphate

extinction coefficient

fructose 1,6-diph08phate

glyceraldehyde 3-ph08phate

guanosine 5'-triph08phate

guanidine hydrochloride

dissociation constant

geometrical average dissociation constant

weight-average molecular weight

weight-average molecular weight extrapolated
to zero protein concentration

weight-average molecular weight determined
from the Svedberg equation using sedimentation
and diffusion coefficients extrapolated to
zero protein concentration

‘g-average molecular weight

‘g-average molecular weight extrapolated to zero
protein concentration

NADH

PEP
PK

520,w

E20,w

Tris

nicotinamide adenine dinucleotide, reduced
form

Hill slepe

phOSphoenolpyruvate

pyruvate kinase

Svedberg, unit of sedimentation velocity

sedimentation coefficient corrected to 20°
and water

sedimentation coefficient corrected to 20°
and water and extrapolated to zero protein
concentration

tris(hydroxymethy1)am1no methane

xi

INTRODUCTION

An interest in the characteristics of yeast pyruvate
kinase was stimulated by the rather convincing evidence
(Rommes, 1964; Pye and Eddy, 1965; Hess and Brand, 1965)
that the pyruvate kinase reaction was rate limiting in both
whole yeast and yeast extracts, and that the enzyme func-
tioned as a control point in the glycolytic scheme. The
successful purification of a stable homogeneous preparation
of the enzyme (Haeckel.g£‘gl.. 1968; Hunsley and Suelter,
1969a) has enabled initiation of studies of the kinetic and
physical preperties of this protein.

The elucidation of the kinetic properties of yeast
pyruvate kinase (Hunsley and Suelter, 1969b) has revealed
marked differences between the kinetics of this enzyme and
the kinetics of the preparation from rabbit muscle (Reynard
gfi'gl.. 1961). While the muscle enzyme exhibits strictly
Michaelis-Menten kinetics with reSpect to all substrates
and cofactdrs, almost the complete opposite is true for the
yeast enzyme. The latter not only possesses homotropic
c00perativity for the substrate phOSphoenolpyruvate as well
as the monovalent and divalent required cations, but is also
markedly activated heterotropically by fructose 1,6-diphos-
phate in a feed-forward fashion, an observation which is
consistent with the postulated role for the enzyme in

1

2
glycolytic control of yeast metabolism. Fructose 1,6-diphos-
phate apparently has no Specific effects on muscle pyruvate
kinase. The only apparent kinetic similarity between the
two enzymes, in fact, is their requirement for monovalent
and divalent cations as cofactors.

It was our intention to examine the physical proper—
ties and conformational changes of yeast pyruvate kinase
using some of the techniques as they were applied to muscle
pyruvate kinase (3.5.. Suelter, 1967; Kayne and Suelter,
1968) with the hepe that the similarities or differences
revealed by such studies would lead to insights into not
only the mechanism of enzyme action, but also the role of
conformational transitions and subunit interactions in
ligand-induced c00perative effects. In addition, the marked
instability of the yeast enzyme, as first described by
Washio and Mano (1960) and the role of glycerol in stabiliz-
ing the enzyme (Hunsley and Suelter, 1969a) set the stage
for the discovery of two prOperties of yeast pyruvate kinase:
cold lability and fructose 1,6-diphoephate-induced instabil-
ity, which will be described in this communication. Consider-
able advantage was taken of these preperties in elucidating
the structural and conformational characteristics of the
enzyme.

Preliminary reports of this work have been presented
(Kuczenski and Suelter, 1970a,b; Kuczenski and Suelter,
1970c,d, submitted to Biochemistry for publication).

IJTERATURE SURVEY

It has been a commonly accepted and well-warranted
procedure in enzymology to attribute to purified enzymes a
greater stability at temperatures lowered to near 0°.
Likewise, stabilization has been attempted (and frequently
achieved) by the addition of substrates and/or Specific
metabolic effectors of the enzyme. The assumption that
these effects might be mediated by conformational transi-
tions of the protein implies the existence of conformations
of varying degrees of stability. However, since there are
no theoretical grounds for suggesting that conformations at
0° or in the presence of Specific metabolites must be the
most stable, it seems only logical to assume that, for some
enzymes at least, low temperatures or the presence of sub-
strates could induce less stable conformers. The fact,
though, that Grisolia, in publishing a review on substrate-
induced inactivations in 1964, was prompted to acknowledge
his indebtedness to editors and their antagonism "... for
having thus forced (him) to further prove this point," might
be favorably or unfavorably interpreted as revealing a
tendency of scientists to adopt a rather zealous "show me"
attitude.

Nevertheless, the observations of Grisolia concern-

ing substrate-induced instability have been convincingly
3

L.
documented in recent literature. In addition, an increas-
ingly large number of reports have been appearing which
support the contention that lowered temperatures may be
detrimental to the vitality of isolated enzymes. In this
review, we will be concerned firstly and primarily with
those enzymes which are cold labile, and secondly those
enzymes which experience cold lability induced by substrates
or effectors. The decision to limit the review in the
latter case, was made not only because of particular rele-
vance to the subject of this thesis, but more importantly
because a surprisingly large percentage of substrate-
destabilized enzymes described in the recent literature fall
into this category. The formal division of the review into
two sectionedis also arbitrary, and considerable overlap

will be apparent.
Cold Lability

One of the earliest discoveries of a cold labile
enzyme1 was reported by Hofstee (1949) in describing the
preperties of Jack bean urease. He attributed the loss in
activity to an enzyme aggregation on the basis of indirect
evidence, however he did not pursue the matter further.

It was not until the early 1960's that other reports

of cold labile enzymes began to appear, with the number since

 

1The term "cold labile enzyme" is used throughout
this thesis to refer to those enzymes which eXperience
enhanced instability at temperatures lower than room tem-
perature or normal physiological temperature.

5

then increasing steadily. In 1960, Shukuya and Schwert

described the purification and prOpertieS of glutamic decar-

boxylase from E. coli. Dilute solutions of the enzyme were

more stable at 20° than at 0°.
albumin protected against the inactivation, while pyridoxal

Addition of bovine serum

phOSphate, a required cofactor, not only protected but also

reversed the inactivation. Recent electron micrOSCOpic

data (Tikhonenko g}; 22., 1968) has revealed that low temper—

ature incubations yield a disaggregation of the polymeric

enzyme from its native state. Strausbauch and Fischer (1970)

have determined from sedimentation data that the enzyme is a
hexamer which participates in a dissociation-association

eQuilibrium. They suggested that the hexamer is the active

Species, and that conditions which lead to dissociation might

’18 1d inactivation, as seen with dilute solutions of the

el'lzyme at 0°. Similarly, dilute solutions of D(-)B-hydroxy

butyric acid dehydrogenase were highly unstable at 0°
A partial restoration of

(>5311118ter and Doudoroff, 1962).
The stability of

act 1vity could be obtained by rewarming.
the enzyme increased at increased protein concentrations, as
did. the reactivation, suggesting an equilibrium which the

a"“l‘t‘ol‘iors considered might involve a dissociated enzyme Species.

In 1963, Dua and Burris successfully purified and

Btabllued a Nz-fixing enzyme from Clostridium pasteurianum

‘hlch exhibited a maximum stability at 22°. Inactivation.

whiCh was biphasic, was again inversely proportional to

131‘Otein concentration, and partial reactivation could be

6

accomplished by rewarming. The extent of the reactivation,

however, decreased as the incubation time at 0° increased,
Suggesting both reversible and irreversible inactivation

steps. The authors attempted stabilization of the enzyme

by the addition of 0.1 M glycerol, but were unsuccessful.
Reference was made to an apparent stabilization by 50%
(v/v) glycerol of a yeast adenosinetriphOSphatase (Meyerhof

and Ohlmeyer, 1952). Although the latter authors did. not

recognize this enzyme's cold lability (as will be seen
later), their use of high concentrations of glycerol to
stabilize the ATPase was similar to the use by Lovelock and
Bishop (1959) of glycerol, glucose, and dimethylsulfoxide to
Protect red blood cells and Spermatozoa against freezing
damage, and to the use of glycerol to stabilize catalase
against freezing damage (Shikama and Yamazaki, 1961) .
Backer and coworkers (1963) pointed out that the beef

heart mitochondrial ATPase was also stabilized by 20-50%
gl5»'c:erol, after having shown that the yeast enzyme, like
the beef heart enzyme (Pullman gt _a_l.. 1960) was cold
labile. In an effort to eXplore the nature of the inactiva-
tion, Penefsky and Warner (1965) examined the effect of
temperature on the sedimentation coefficient of the enzyme.
At 25° the ATPase sedimented as a single peak with £30m =
12.9 8, while incubation of the enzyme at 0° for 5 hr yielded
a mixture of species with sedimentation coefficients of 3.5 5

an 9.1 8. The amount of 3.5 8 Species was inversely propor-

ticDual to protein concentration. Both activity and the 12.9 S

7
Species could be recovered by warming the sample to 25°.
0n the other hand, glycogen phOSphorylase b appar—
ently undergoes a much more complex inactivation mechanism
(Graves gtflgl.. 1965). The sedimentation coefficient of
the enzyme increased from 8.4 S to 20.2 S as the inactiva-
tion progressed. Rewarming in the presence of pyridoxal
phosphate gave complete reactivation only at protein con-
centrations greater than 0.1 mg/ml. Not only the extent,
but also the initial rate of inactivation were dependent on
the first power of protein. At the same time, however, the
course of the inactivation followed pseudo-first-order
kinetics to a lesser extent as protein concentration was
increased, and the rate of inactivation then rapidly decreased.
As a result, the enzyme, as measured by its half—life, became
apparently more stable as protein concentration increased,
a dependence on protein opposite to the dependence on protein
«of the initial rate of inactivation. The authors prOposed a
Inechanism of inactivation involving conversion of the native
eunzyme to an inactive Species and formation of an inactive
Species-native enzyme complex which was no longer cold
1£Ibile, to account for the protein concentration effects.
Both the observation that fully inactivated phOSphorylase b
aK1ded.to active enzyme has no effect on the rate of inacti-
‘Vlrtion, as well as the requirements for reactivation
‘16 scribed above, however, seem incompatible with the absence,
1n.the proposed mechanism, of a dissociated enzyme Species

as an intermediate during the inactivation.

8

An equally complex low temperature inactivation
phenomenon has been described for 17-B-hydroxysteroid
dehydrogenase from human placenta (Jarabak £2 21., 1966).
The enzyme undergoes a biphasic, pseudo-first-order inacti—
vation which depends inversely on protein concentration.
However, the inactivated product, which on polyacrylamide
moves as a mixture of defined. Species more slowly than the
native active Species, is eluted from Sephadex G-100 with
the void volume. Rewarming partially reverses the inacti-
vation only on protein which elutes with the native enzyme
or after the native enzyme. The mechanism appears to
involve a reversible dissociation, followed by an irrever—
Sible aggregation. 50% glycerol completely stabilizes, and
1 M P03- or 10 uM substrate or inhibitor (diethylstilbesterol)
partially stabilize.

Steer liver arginosuccinase (Havir st 21.. 1965)
apparently inactivates independent of protein concentration,
tit 0°, although the authors only examined concentrations of
1 and 6 mg/ml. The inactivation is two-step, however, and
Only the first step can be reversed by rewarming. While
'tkie enzyme at low ionic strength is cold labile, the addi-
tion of Tris buffer enhances the inactivation, whereas
potassium phOSphate stabilizes.

Pyruvate carboxylase from chicken liver mitochondria
<¥1ear1y undergoes a dissociation during cold inactivation
(Scrutton and Utter, 1965). The sedimentation coefficient

Changes from 15 S to 7 S at low temperatures, but, as the

3L1

00

pa

9

authors pointed out, the amount of 7 S material was not
proportional to the extent of inactivation. Addition of
1.5 M sucrose [or 2.5 M methanol (Irias 23 21.. 1969)]
provided complete stabilization. Acetyl CoA, a required
cofactor, also stabilized. The inactivation can be only
partially reversed by rewarming in the presence of ATP.
Irias 22,2}. (1969), in further describing the cold labil-
ity of the enzyme, characterized the inactivation as con-
sisting of two pseudo-first-order steps in which the
second step was irreversible. Low concentrations of urea
at 23° appear to mimic the effects of low temperatures, and
acetyl CcA protects against this inactivation as well. The
data are consistent with some dissociation phenomenon, and
it could be argued that the 7 8 Species described by Scrutton
and Utter arises from the irreversible inactivation step.

Several other cold labile enzymes have been described
:recently. These include chicken liver fatty acid synthetase
(Hsu and Yun, 1970) , and UDP-galactose 4-epimerase from
bovine mammary gland (Tsai gt g._l_. . 1970). Both enzymes are
completely stabilized by glycerol at 0° (20% and 50% reSpec-
‘tisvely). Interestingly, the latter enzyme at 25° is
destabilized by glycerol. Lee and Muench (1970) have
described an interesting cold labile enzyme from E. _<_:_c_>_l__i_.
The prolyl tRNA synthetase, which must be purified in the
Presence of 40% glycerol to retain activity, participates
in an active dimer-inactive monomer equilibrium. At 0°, in

the absence of glycerol, the inactive monomer predominates,

10
and on storage, this Species irreversibly inactivates. Con-
version of the monomer to dimer can be obtained either by
incubation at 37° in the absence of glycerol, or at 0° in
the presence of glycerol.

Several characteristics of cold labile enzymes can be
summarized. Polyhydroxic compounds such as glycerol appear
to protect against low temperature inactivation for all
those enzymes which have been examined from this point of
view. That Bus and Burris (1963) saw no such effect with
glycerol may indicate that they did not use sufficiently
high concentrations (only 0.1 M) of the polyhydroxic compound.
In addition, a dissociation step in the mechanism can be
implicated for most cold labile enzymes. At least as early
as 1955, von Hippel and Waugh described a low temperature-
1nduced protein dissociation involving casein, which could
be reversed by rewarming the mixture to 32°. Most of the
<3nzymes described in this review could be at least partially
reversed by rewarming. Only glycogen phOSphorylase b, and

possibly urease appeared to undergo aggregation reactions
111 the functional inactivation step, but, in neither case
was the data conclusive. Finally, the inactivation as
measured as a function of time appears, in most cases, to
rOllow biphasic pseudo-first-order kinetics, in which the
Second step is frequently irreversible. As will be described
111 this thesis, yeast pyruvate kinase is typical as a cold
lalflde enzyme with reSpect to most of these phenomena.

11

Metabolite-Induced Inactivation

AS mentioned previously, Grisolia reviewed the sub-
Ject of substrate-mediated destabilization in 1964. A
considerable impetus was given to writing such a review
from experiments in his laboratory with frog liver carbamoyl
phOSphate synthetase. The unexpected observation was made
that acetyl glutamate, a required cofactor, in the presence
or absence of Mg2+, or the substrate ATP in the absence of
Mg2+ led to a marked instability of the enzyme (Caravaca
and Grisolia, 1959), while ATP plus Mg2+ stabilized. In a
later communication (Raijman and Grisolia, 1961), the
authors showed this inactivation to be much more rapid at
lowered temperatures.

Recently, Guthhhrlein and Knappe (1968) examined the
same enzyme from rat liver in more detail. The enzyme,
Which is stabilized by 20% glycerol, undergoes a Slow acti-
‘VStion.phenomenon in the catalytic assay at temperatures
lmslow 15°. The rate and extent of activation are propor—
tilonal to the acetyl glutamate concentration and are first
erder. If the enzyme is stored at 0°, then assayed at 10°.
the activation kinetics are biphasic, from which the authors
8uggested the existence of two different inactive Species,
11 and I2. Prolonged storage at 0° resulted in a slow
irreversible inactivation. Addition of acetyl glutamate at
temperatures above 25° converts the enzyme to the fully

active Species, whereas the same ligand at 0°, in the absence

12
of substrates, yields complete conversion of the enzyme to
the more slowly-activated inactive Species (I2). This con-
version to I2 is directly proportional to acetyl glutamate
concentration. Sedimentation data indicated that the active
enzyme and 11 were similar in molecular weight, whereas I2
was markedly reduced in size. Hence, the same ligand,
depending on the temperature and the presence of substrates,
could lead to either activation, or to an inactive conformer
which readily dissociated at the lower temperatures.

The pyruvate carboxylase system described in the
previous section (Irias 35 31., 1969) also involves a
substrate-enhanced inactivation. Acetyl CoA alone or in
conJunction with ATP, Mg2+, H003 and pyruvate stabilizes the
enzyme. Pyruvate alone, however, produces an enhanced low
temperature inactivation.

Threonine deaminase in crude extracts of R. subtilis
is cold labile in the presence of the cofactor pyridoxal
phOSphate (Hatfield and Umbarger, 1970). It is also

destabilized by the same ligand at 37°, but the ligand
c<>mpletely stabilizes at 22°. Treatment of the crude
elttract with CaPOu gel eliminates the cold lability and
Tpflroduces the unusual effect of inducing a room temperature
lérbility. The authors did not pursue this phenomenon.

Yeast glyceraldehyde 3-ph08phate dehydrogenase is
stable at 0°, but addition of ATP produces a rapid inactiva-
'tlon, accompanied by a complete dissociation of the enzyme

to subunits (Stancel and Deal, 1969). The subunits have

A J.

.'l

(J'

‘3’

’71

13
been described by the authors as "folded," because of their
relatively high sedimentation coefficient. Complete reasso-
ciation may be achieved by rewarming only in the presence of
sucrose and the dissociating agent, ATP. 0n the basis of
other data (Yang and Deal, 1969) the previous authors sug-
gested that the rapid conformational transition which later
results in the dissociation was the physiologically signifi-
cant effect of ATP. The enzyme thus becomes more susceptible
to proteolytic digestion.

Similarly, Rosen.g£,§l.. (1967) observed that
Dafructose 1,6-diphOSphatase, which is inhibited by AMP, is
dissociated by the ligand at pH 9.2 only at low temperatures;
the substrate, FDP. protects against this dissociation. If
the enzyme is desensitized chemically to AMP inhibition, it
is no longer cold labile in the presence of the inhibitor.
{The authors describe the effect as an "... extreme manifes—
tation of more subtle alterations ... which occur in the
presence of substrate at physiological pH."

Glutamate dehydrogenase provides an interesting final
eIample of substrate induced instability. In 1957, Fincham
‘18 scribed a mutant of NeurOSpora crassa which did not grow
Eit: 20°, but did at temperatures above 25°. The mutant
EKJLWutamate dehydrogenase was reversibly inactivated at 21° or
below, but was stable at higher temperatures.

Eisenkraft gt‘gl.. (1969) later showed that the gluta—
mate dehydrogenase from beef liver had to be purified in the

Presence of 50% glycerol to maintain stability. Concentrations

14
of NADH less than 10 cm destabilized the enzyme, whereas
concentrations above 10 uM stabilized it. The inactivation
was reversible (proportional in extent to the activity
remaining) by the addition of ADP. Inactivation kinetics
were pseudo-first-order, and the rate was directly prepor-
tional to the concentration of a 310,000 molecular weight
"monomer." Since the enzyme participates in an equilibrium
with a dimeric form, the authors suggested that the dimer
was a stable Species under the conditions described, and
that the monomer was the "active Species in the inactivation."
The irreversible step in the inactivation apparently involves
a dissociation of the monomer to a lower molecular weight
Species. Henderson and Henderson (1969) further character-
ized the inactivation using GTP, an inhibitor of the enzyme,
as the inactivating ligand. In D20, the enzyme is stable at
low temperatures, and GTP no longer inactivates. But,
kinetically, GTP acts as a stronger inhibitor in D20 than in
320. D20 also prevents the NADH-induced dissociation. The
authors interpreted the results in terms of the monomer-
dimer equilibrium discussed above, suggesting that if the
dimer were the active Species, and GTP favored the inactive
monomer, then the monomer would appear to be an intermediate
in the irreversible dissociation to subunits promoted by
both GTP and NADH. If D20 favored the inactive monomer over
both the active dimer and the subunits, it would not only
stabilize against irreversible dissociation, but also
increase the inhibitory effect of GTP in the catalytic assay.

15

An.1nduced inactivation promoted by either low tem-
peratures or Specific ligands, or the combination of the
two, can thus provide data concerning the structural
properties of enzymes. An inactivation, like a stabiliza-
tion, promoted by a ligand which has been observed to affect
the kinetics of an enzyme-catalyzed reaction, is direct
evidence for a conformational transition resulting from the
interaction with the ligand. Since internal bonds between
regions of polypeptide chains or between subunits of a
polymeric protein are implied to be weakened or strengthened
during a destabilization or a stabilization, effects of
temperature or solvents such as glycerol or D20 on the
secondary effects of ligand-protein interactions can pro-
vide insights into the mechanism by which such interactions
are mediated. Although the evidence is not conclusive,
physico-chemical and thermodynamic data are available for
interpretation of the nature of those bonds which can be
affected at low temperatures or in the solvent systems

described above, and will be discussed later.

METHODS AND MATERIALS

Lang-mes

Pyruvate kinase was isolated from fresh "Budweiser"
bakers' yeast (Anheuser-Busch, Inc.), Saccharomyces
cerevisiae, according to the procedure of Hunsley and
Suelter, (1969a). The enzyme was stored as a concentrated
suSpension at 4° in 90% saturated (3.6 M) (N34)2804. The
minimum Specific activity of any preparation used in these
studies was 210 umoles/min per mg. Prior to use, the enzyme
was chromatographed at room temperature on a column of
Sephadex G—25 (coarse). Aliquots were tested with saturated
BaClz to ensure them free of ammonium sulfate.

Lactic dehydrogenase used in the coupled assay system
was the Sigma type II rabbit muscle enzyme, and was desalted
free of (NH4)2804 using a column of Sephadex G-25, when
cation content of the assay was to be critically controlled.
Ammonium sulfate suSpensions of rabbit muscle aldolase were
Sigma products, and rabbit muscle d-glycerophOSphate dehydro-
genase-triose phoSphate isomerase mixed crystals were
Calbiochem products. Rabbit muscle pyruvate kinase was
isolated from frozen.rabbit muscle (Pal—Freeze Biologicals)
by a modification (Kayne and Suelter, 1965) of the procedure
of Tietz and Ochoa (1958).

16

17
II. Chemicals

All water was either double glass distilled or glass
distilled and deionized (Crystalab Deeminite). (CHA)4FDP.
(CHA)3PEP, NaADP, NaNADH, Dimethyl Di-(CHA) form of DHAP,
arfl.the diethyl acetal.Ba form of G-3-P were Sigma products.
NaADP was converted to the Tris salt by treatment with
iDcwex 50WAX8 in the Tris form. (CH3)4NC1 from Aldrich was
recrystallized from absolute ethanol and passed over a
column of Chelex 100 in the Tris form to remove contaminating
heavy metals. Maleic anhydride was also an Aldrich product.
GuHCl was either Mann.Ultra.Pure and used directly, or was
obtained from.Eastman as the carbonate and converted to the
hydrochloride according to the procedure of Kawahara 32 21..
(1965). Mercaptoethanol (Sigma) was redistilled before use.
All other chemicals and reagents were used without further

purification.
III. Experimental Methods

1. Assay of activity and determination of enzyme concen-

tration

Yeast pyruvate kinase concentration was estimated

from the absorbsnce at 280 nm.(§2'§:

Suelter, 1969a). Standard assays were performed at 30° by

= 0.653) (Hunsley and

employing the linked lactic acid dehydrogenase assay modified
from Bacher and Pfleiderer (1955). The reaction mixture con-
tained in 1 m1: 100 umoles (CH3)4N cacodylate, pH 6.2; 24

18
umolos MgClz; 100 umoles KCl; 10 umoles (CHA)3PEP; 10 umoles
NeADP. pH 7.0; 1.0 umoles (CHA),+FDP; 0.15 umoles NaNADH; and
33 ug lactic dehydrogenase (Hunsley and Suelter, 1969b).
Aliquots of enzyme were added to the reaction mixture at 300
in.a 1 cm Silica cuvette to initiate the reaction, and the
change in Optical density at 340 nm was recorded on a Gilford
model 2000 modified Beckman DU ultraviolet SpectrOphotometer.
The initial rate was converted to micromoles of pyruvate
formed per minute using the extinction coefficient for NADH
(Horecker and Kornberg, 1948).

ADP and PEP concentrations were estimated by a modi-
fication of the Bucher and Pfleiderer (1955) pyruvate kinase
assay in the presence of excess rabbit muscle PK. FDP was
estimated in the presence of excess aldolase as modified from
the assay of Rutter gt a}. (1966).

2. Ultracentrifugal analyses

A Spinco Model E analytical ultracentrifuge equipped
with phase-plate schlieren optics and Rayleigh interference
Optics, and an RTIC unit was used for all ultracentrifugal
experiments. Sedimentation velocity eXperiments were run at
59,780 rpm usingamodel AnD rotor, or at 50,760 rpm with an
AnE rotor. Diffusion coefficient eXperiments were performed
in double-sector capillary-type synthetic boundary cells at
4908 rpm and the coefficients were calculated using height—
to-area analysis (Schachman, 1957).

A molecular weight for native enzyme was also deter-

mined using the meniscus depletion technique of thantis

19
(1964). Runs were performed at 20° with a rotor Speed of
15,200 rpm using Rayleigh interference optics and the six-
channel Kel-F centerpiece designed by thantis (1964).
Rayleigh patterns were recorded on Kodak II-G photographic
plates.

The short-column sedimentation equilibrium technique
cf'Van fields and Baldwin (1958) was used for molecular
weight determinations in 6 M GuHCl. These SXperiments were
run for 30 hr (results remained constant from 30 to 40 hr)
near 20° with a solution column depth of 1.7 mm. Enzyme
for these SXperiments was prepared by extensive dialysis
(48 hr) against the apprOpriate GuHCl solution at 4°.

A molecular weight for the subunits of yeast PK was
also determined with maleylated enzyme. A modification of
the procedure of Freedman gt a}. (1968) was used to prepare
maleylated yeast PK. Stock enzyme (20 mg) in 1.0 ml was
dialyzed extensively against 0.05 M sodium borate buffer,
pH 9.0, at 0°. Maleic anhydride in acetone (100 pl of a
0.5 g/ml solution) was added to this solution in 10-ul
aliquots over a 30-min period. pH 9 was maintained with
5 N NaOH utilizing a Radiometer TTT-i-SBRZ-SBUl-TTA31
automatic recording tetrator. The protein was then dialyzed
for 2 days against several changes of 0.1 M KCl-0.1 M potas-
sium borate, pH 9.0.

Densities of solutions were determined by pyncnometry.
Viscosities of GuRCl solutions were interpolated from data

of'.Kawahara and Tanford (1966). All other Viscosities were

20

based on data from Bates and Baxter (1929), or from Svedberg
and Pederson (1940).

Calculations, including statistical analyses of the
data, were performed on a Control Data Corporation 3600
digital computer using fully tested programs [sedimentation
velocity, diffusion coefficients, and low—Speed equilibrium.
(W. C. Deal, Jr.. in preparation); high Speed equilibrium,
with a modification of a program from Small and Resnick
(1965)].

3. Fluorescence analyses

Fluorescence measurements were made with an Aminco-
Bowman SpectrOphotofluorometer equipped with an X-Y recorder.
Fluorescence at right angle to the illumination was measured
with the indicated temperature maintained using a constant
temperature accessory. Several determinations were checked
using front-face illumination and emission, but this tech—
nique was discarded since results were identical to those
obtained using right angle illumination and emission.

Titrations of fluorescence change were routinely
begun with an initial volume of 2.0 ml of sample in a fused
quartz cell (capacity 4.8 ml). Small aliquots of the titrant,
which was contained in a solution of identical composition as
the sample (including protein concentration) were added and

mixed with a magnetic stirrer.

RESULTS
I. Temperature Stability of Yeast Pyruvate Kinase

Figure 1 Shows the effect of temperature on the sta-
bility of yeast pyruvate kinase at 0.5 ma/ml in 0.1 M
Tris-RC1, pH 7.5. Over a period of 72 hr less than 10% of
the activity was lost at 23° whereas at 0° over 95% of the
activity was lost over the same time interval. Each point
represents an assay of an aliquot of enzyme diluted directly
into the assay mix at 30°. Assays were linear over the 1-3
min observation period.

Before each stability study, but after treatment with
Sephadex, the protein was allowed to stand at 23° for 3 hr.
Little or no change in Specific activity could be observed
over this time period. This preincubation step was intro-
duced in this and all future eXperiments to eliminate the
unusual results obtained without preincubation (Figure 2).
Preincubation of the enzyme at 23° for 3 hr eliminates the
initial activation observed at 0.05 mg/ml (lower curves),
but not at 0.5 mg/ml (upper curves). Both samples were
eluted from the Sephadex column at about 0.6 mg/ml. The
activation observed at low protein concentration was
Qualitatively reproducible, and was not observed above 0.10

Ina/m1. The activation was observed when dilution to 0.05 mg/ml

21

22

Figure 1. Effect of Temperature on the Stability of Yeast
Pyruvate Kinase

Protein was diluted to 0.5 mg/ml after chromatography
on Sephadex in 0.1 M Tris-HCl, pH 7.5. Each point represents

an assay of an aliquot of enzyme removed from the incubation

mixture.

Specific Activity

250
200

IOO

(D
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A
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Time (hrs)

24

Figure 2. Effect of Preincubation at 23° on the Stability
of Yeast Pyruvate Kinase at 0°

Closed circles represent enzyme Sephadexed into 0.1 h
Tris-RC1, pH 7.5 at 23°, then immediately diluted to the
indicated protein concentration at 0°. Open circles repre—
sent enzyme treated with Sephadex and allowed to stand for

3 hr. at 23°, then diluted to the final concentration at 0°.

so
60
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26
was made at 23 or 0° either from Sephadex-chromatographed
stock solution or from ammonium sulfate precipitate, and
either into a glass or polyprOpylene reaction vessel. The
23° dilution, however, resulted in a higher activation over
a much shorter period of time.

To determine whether any changes in the sedimentation
pattern of the enzyme could be observed as a result of the
preincubation period, aliquots of the ammonium sulfate pre-
cipitate were treated in two ways. One was chromatographed
over Sephadex into Tris buffer, diluted to 6.0 mg/ml, and
allowed to stand at room temperature for 3 hr. The second
was centrifuged in the preparatory centrifuge for 10 min at
0°, the precipitate was collected, and at the end of the
3-hr preincubation period for the former sample, was dis-
solved in 0.1 M Tris buffer. Both samples were immediately
centrifuged at 20°. The sample preincubated after treatment
with Sephadex sedimented as a single symmetrical peak (8.0 S),
while the other contained three peaks with approximate g
values of 7.7. 11.5. and 13.6 S, in a ratio of approximately
“:3:3.

II. Molecular Weight of Yeast Pyruvate Kinase

1. Native enzyme
1. ,gflg molecular weights
Initial attempts to determine the moleculae weight of
the native enzyme were performed in 0.1 M KCl, 2.6 x 10"2 M
MgCle, 10-3 M (CHA)4FDP, and 0.1 M (CH3)uN cacodylate buffer,

27
pH 6.2, the conditions for optimal catalytic activity. The
sedimentation constant in this solvent, found by extrapola-
tion to zero protein concentration.(ggo’w) was 8.85 S
(Figure 3). In a Similar manner, the diffusion coefficients
extrapolated to zero protein gave a value of 2§0,w = 4.84 x
10-7 cmZ/sec (Figure 4). These values, together with a
partial Specific volume of 0.734 cc/g as calculated from the
amino acid content (Hunsley and Suelter, 1969a) using the
procedure of McMeekin and Marshall (1952), yielded a weight
average molecular weight (M3(§[Q)) of 166,500.

During the above eXperiments, a Slow precipitation of
protein, particularly at higher protein concentrations, was
observed. This problem was eliminated in 0.1 M Tris, pH 7.5.
and thus the studies were repeated in this solvent. Values
of §30.' = 8.34 S (Figure 5), and_I_)f2’0.W = 4.52 x 10"7 cm2/sec
(Figure 6) yielded a molecular weight of M3(§/_D) = 168,100.
The diffusion coefficient was determined both after a 3 hr
preincubation, and after an 18 hr preincubation. Varying
the time of preincubation after 3 hr had no effect on the
sedimentation pattern of the enzyme.

11. High Speed equilibrium technique

The molecular weights of the native enzyme as deter—
mined by the meniscus depletion technique are plotted in
Figure 7 as number-average molecular weight zg. concentra-
tion in fringes for enzyme in 0.1 M Tris-H01, pH 7.5. con-
taining 0.23 M KCl, 2.5 x 10-2 M Mg012, 2 x 10-3 M FDP, and
10-2 M PEP. The enzyme retained 92% of its initial activity

28

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38
when allowed to stand for 24 hr at 20° at 0.2 mg/ml in this
solvent. The enzyme was not sufficiently stable for a 24-hr
period at 0.2 mg/ml to determine the molecular weight in the
absence of FDP.
2. Subunits

1. Enzyme in guanidine hydrochloride

Preliminary sedimentation equilibrium eXperiments
with yeast PK in 6 M GuHCl indicated the need for a high
concentration of reducing agent to eliminate what appeared
to be a random aggregation of the protein. Figure 8 presents
data, plotted according to Van Holde and Baldwin (1958), from
a typical sedimentation equilibrium experiment with yeast PK
in 6 M GuHCl containing 0.15 M Z-mercaptoethanol. Data for
‘M‘ and}!z of yeast PK at several concentrations are plotted
and extrapolated to zero protein concentration in.Figure 9.
Values for M: and M; were calculated to be 41,400 and 45,900.

reSpectively. Under the same solvent conditions, the enzyme

0.8%

sedimented as a single symmetrical peak (£20 w
I

= 1.09 s).

11. Maleylated enzyme

To further characterize the subunits, efforts were
made to dissociate the enzyme through the introduction of
negative charges by maleylation. Under the conditions
described in Methods, 230 moles of maleate/168,000 g of
protein (11.9% of the resultant molecular weight) were
incorporated as determined by the SpectrOphotometric assay
of Freedman gt 3.}. (1968). Extensive dialysis of the

maleylated enzyme did not alter the absorption coefficient

39

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43
of the enzyme at 250 nm, suggesting complete removal of
unbound maleate.
Results for the sedimentation coefficient and the

diffusion coefficient of the maleylated enzyme determined
as a function of protein concentration are shown in
Figures 10 and 11 reSpectively. The values of Ego,w =
1.97 S and 230." = 3.75 x 10'7 cmZ/sec yielded a molecular
weight of 53(5/2) = u?,9oo. Subtracting 5700 for the con-
tribution of the bound maleyl groups gave a net molecular
weight of #2,200 for the subunits. Addition of 0.1 M
Z-mercaptoethanol to the maleylated enzyme affected neither
the sedimentation coefficient nor the diffusion coefficient.
The sedimentation and diffusion coefficients of yeast PK
under all of the conditions described above are summarized
in Table I.

3. Effects of cations, substrates, and FDP on the sedi-
mentation coefficient of yeast pyruvate kinase

Although the results presented in Figure 7 suggest

that no associationpdissociation equilibrium involving
interaction of the enzyme with ligands is apparent, the
effect of cations and other ligands, separately and together,
on the sedimentation coefficient of yeast PK was measured to
further rule out that possibility. The results are presented
in Table II. The only changes 1n-§80,w occur in the presence
of FDP with no K+ (EIpt. h), and in the presence of FDP, K+,
and PEP (Expt. 5), both with Mgz+ present. In both cases,

slight increases in_s_§o’w are obtained. As will be seen

1+4

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48

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ommsam mpm>zamm ammo» go mesmdodmgooo modpmpsmafipom

HH mqmde

50
later, the concentration of FDP used in EXpt. 3 in the
presence of K+ was not saturating so that the results

obtained for that eXperiment are not conclusive.

III. Effects of Substrates, Cations and FDP on the

Fluorescence of Yeast Pyruvate Kinase

1. Qualitative effects
The fluorescence of yeast PK is quenched by the addi-

tion of the cations K+ and/or Mgz+

, but not (CH3)4N+. How-
ever, as can be seen from Table III, the extent of the
quenching produced by these cations individually or together
is minimal when compared to the effect on the fluorescence
of yeast PK brought about by the addition of FDP. The extent
of the quenching produced by FDP was independent of cations
and/or PEP, although, as will be seen, the concentration of
FDP required to bring about the total fluorescence change
depends on the effectors present. Because of the instabil-
ity of yeast PK in the presence of FDP alone, a quantitative
determination of the interaction of FDP in the absence of
cations with the enzyme could not be made, although it can
be stated that the fluorescence quenching by thisligand
,/#““~~m~w~dMM/’ I
under these conditions is significantly greater than theV
quenching obtained in the presence of only the cations; The
upper curve in Figure 12 represents the fluorescence emis-
sion spectrum of yeast PK at 0.30 mg/ml in 0.1 M Tris-H01,
pH 7.5, excited at 280 nm. Addition of 5 x 10-3 M FDP and

2.5 x 10-2 M Mg012 reduced the intensity at 3nd nm by 12%,

51

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.ag 0mm was sewsoa

 

 

 

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52

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54
and shifted the peak position slightly to 347 nm.
2. Titration of yeast pyruvate kinase with FDP by

measurement of fluorescence change

Since the changes in the fluorescence of PK after
addition of FDP were sufficiently large to allow for a
determination of dissociation constants from titrations of
the fluorescence change, we proceeded to examine the extent
of interaction of the enzyme with cations and substrates as
reflected in the binding of FDP. Except as noted, the
excitation wavelength was 280 nm and the emission was
monitored at 344 nm. The data were treated as shown in
Figure 13 as a Hill plot (Atkinson, 1966) of the fluores-
cence of PK as a function of the concentration of FDP at
various KCl concentrations. As can be seen from Figure 13
and Table IV, the addition of 0.10 M KCl (the optimal KCl
concentration for catalytic activity of PK in the presence
of FDP) increased the apparent KD for FDP from 0.09 mM to
1.3 mM with little effect on.the Hill lepe (2H)' Increas-
ing the KCl to 0.23 M (Optimal for catalytic activity in
the absence of FDP) increased the apparent KD for FDP even
further to 3.1 mM. 0n the other hand, addition of 0.01 M
PEP at the latter KCl concentration markedly increased the
affinity of the enzyme for FDP yielding an apparent KD of
0.069 mM. It should be noted that identical KD'S and nH's
were obtained when fluorescence differences were determined
at 340 to 355 nm, and when the excitation wavelength was
280 to 295 nm.

55

Figure 13. Plots of the Binding of FDP to Yeast Pyruvate
Kinase as Monitored by Fluorescence Changes

Protein was maintained at 0.30 mg/ml and the tempera-
ture was 23°. The excitation wavelength was 280 nm. The

number in parentheses is the Hill slepe (2H)°

-I)

AF

 

AFMOX

Log (

56

 

2 I I I T I I I I IA
I. — , / _.
. .1 / .
c. . .—
"0 /(l.72)o
. A
0.3.. (I55),A ,’ /(|.6|) (L37) _.
A l0 o
0.6-- 7] 9 / ..
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A
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0'81: o ‘. 0.0l M Pee
A 0 o /
A
-l.O- o -—
I I° I I I I I I I
2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2

-Log [FDP] (M)

57

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58

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59

In an effort to separate Specific monovalent cation
effects from general ionic strength effects, dissociation
constants for FDP and PK were determined in the presence of
the same concentrations of (CH3)uN+, a catalytically non-
activating cation (Figure 14). The effect of (CH3)4N+ on
the apparent dissociation constant for FDP is analogous to
the effect of K+, although the extent of the increase in
apparent KD is somewhat lessened. 0n the other hand, a
considerable increase in the Hill lepe was obtained with
(CH3)4N+ which was not observed with K+. In addition, the
combined presence of PEP and (CH3)1,N+ does not yield the
marked decrease in apparent KD for FDP as was seen with
0.01 M PEP plus K+. In fact, the apparent KD for FDP in
the presence of 0.01 M PEP with or without (CH3)I+N+ is iden-
tical to the apparent KD for FDP obtained in the absence of
PEP, that is, in the presence of Mg2+ alone, although the
Hill slope is considerably reduced.

The effect of the second substrate of the pyruvate
kinase reaction,.ADP, is also included in Table IV. For
these titrations, an excitation wavelength of 295 nm was
used to minimize absorption due to the ADP. In the
presence of ADP and either K+ or (CH3)4N+ the apparent KD
for FDP is reduced by an order of magnitude from the value
obtained in the absence of ADP; the Hill slope in both cases
is reduced from values near 2 to unity.

The influence of temperature on the quenching of PK

fluorescence by FDP is also included in Table IV. Under

60

Figure 14. Plots of the Binding of FDP to Yeast Pyruvate
Kinase as Monitored by Fluorescence Changes

Protein was maintained at 0.30 mg/ml and the temper-
ature was 23°. The excitation wavelength was 280 nm. The

numbers in parentheses are Hill slepes (nH).

61

 

I I I I T I I I I I
/ -’

|.Or- A ./ ...
(I86) ‘4
A/ 3

(HO)
‘ ._

AA

Additions

 

0.l0 M (CH3)4NC| _.

0.23 M (CH3)4 NCI

0.23 M (CH3)4NCI,1
0.0l M PEP

 

 

 

J J J l l L l l
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0

-Log [FDP] (M)

 

62

each of the three conditions in which effects of temperature
were examined, (no KI. 0.23 M K+, and 0.23 M K+ plus 0.01 M
PEP) the apparent KD for FDP increased by an order of magni-
tude as the temperature was raised from 0 to 30°. However,
no consistent trend for 2H could be seen, with values
decreasing in the absence of K+ as the temperature was
raised from 0 to 30°, and values increasing in the presence
of KI or K+ plus PEP as the temperature was increased from

O to 30°.

IV. Fructose 1,6-Diphosphate—Induced Inactivation of Yeast

Pyruvate Kinase

1. Inactivation at 0°
1. Dependence of the inactivation on FDP concentra-
tion

A search for compounds which might stabilize the
enzyme against cold inactivation revealed that FDP, the allo-
steric activator of yeast PK, markedly enhanced the low tem-
perature inactivation. Efforts were then made to character—
ize the mechanism by which.FDP, in the absence of cations,
promoted inactivation of the enzyme.

Figure 15 shows the effect of temperature on the
inactivation at millimolar concentrations of FDP. The data
in this Figure, as compared to Figure 1 indicate that the
rate constant is increased as much as 1000-fold over the
sample at 23°. (Note that the time scales on these Figures
differ.) To insure that this effect was produced by FDP,

63

Figure 15. Effect of Temperature on the FDP-Enahnced
Inactivation of Yeast Pyruvate Kinase at 0.5
mg/ml in 0.1 M Tris-HCl, pH 7.5
FDP concentration was 1.26 x 10"3 M. Each point

represents an aliquot of enzyme removed from the incubation

mixture and assayed at 30°.

64

 

 

|20 I60 200 240
Time (min)

80

40

 

 

_ 0 _ _ _ _ _ _ _ _
.o
0C \0\ A
D. wwmo We o\A\
m. .... 0x .\. \
T o\ u\ AA
I . on A
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O\ \ \M\A
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03:00 00080

65

PK was incubated at 0° at 1.0 mg/ml under the conditions
shown in Table V. Essentially two rates of inactivation
were obtained: that resembling the 0° curve of Figure 1,
(Samples 1, 2, and 3), and that resembling the 0° curve of
Figure 15 (Samples 4, 5, and 6). The inactivation was not
produced by metal contaminants of the FDP such as cOpper
(Passeron'gtlgl.. 1967) since FDP treated with Chelex and
untreated FDP produced identical results. The dependence
of the inactivation on.FDP concentration was then measured,
and the results are presented in Figure 16, for yeast PK at
0.5 ms/ml.

11. Treatment of the data from Figure 16

The curves obtained from the semi-logarithmic plots
in.Figure 16 are biphasic, indicative of two pseudo-first-
order inactivation steps. Under this assumption, the data
shown in.Figure 16 were treated as in.Figure 17. Extrapola—
tion of the data of the slow step to zero time yielded
intercepts which varied from Specific activity 210-39 umoles/
min per mg as FIP concentration was increased from 0 to 1.26
mM. Identical intercepts were obtained for 0.756 and 1.26
mM FDP concentrations, suggesting that the latter concentra-
tion was saturating. The difference between the intercept
at saturating concentration of FDP and that obtained in the

absence of FDP was equated to unity, and values, defined as

a (intercept)0 FDP - (intercept)X FDP

 

= (1)
(intercept)0 FDP - (intercept)1 26 mM FDP

66

TABLE V

Requirement for FDP for Enhanced Inactivation
of Yeast Pyruvate Kinasea

 

 

 

Additions to a Final Enhanced
Sample Volume of 0.1 ml Inactivation
1 none -
2 1.1 ug aldolase -
3 132 umoles DHAP _

120 umoles GB?
4 132 uncles DEAF +
120 umoles G3P

1.1 mg aldolase

5 126 umoles FDP +
1.1Iw7ffldolase
6 126 umoles FDP +

 

aEach sample contained yeast pyruvate kinase at 1.0 mg/ml.
Samples were incubated at 0°, and activity was followed
with time.

b(-) indicates that resultant inactivation curve resembled
the 0° curve of Figure 1. (+) indicates that inactivation
resembled 0° curve in Figure 15, both in rate of inactiva—
tion, and in extent.

67

Figure 16. Effect of Pb? Concentration on the Stability of
Yeast Pyruvate Pinase at 0° in 0.1 M Tris-HCl,
pH 7.5.

Final protein concentration was 0.50 mg/ml. All

samples had an initial Specific activity of 210 units/mg.

 

N
0

l0

 

 

 

 

 

+ q
u -I
0

v

2

3 756 \. A

e l260 \. o . W)

l J J J L J
40 80 l 20 |60 200 240 28

Time (min)

69

Figure 17. Example of the Method of Treatment of Data from
Figure 16

The sample above was inactivated in the presence of
84 uh F0? in 0.1 M Tris.HCl, pH 7.5 at 0.5 mg/ml of pyruvate
kinase. Inactivation was allowed to continue until the slow
process, labeled 53, became linear. This rate was extrapo-
lated to zero time, and Specific activity values along this
line were subtracted from the eXperimental points (closed
circles). The Open circles represent the differences so

obtained.

7O

 

 

 

 

 

Time (min)

L I I I I I I
200 —
o
0
I00 —‘
3‘ 80 “
E 60 —
0
<1
.9 4O '—
t
o
02
Q
(I)
20 T
o\
\ o
\ k2= 3.9l x I0’2 min’I \
o
\ \4
I ‘ I L I I I
40 80 l20 I60 200 240

and

71
were calculated for each FDP concentration and plotted vs.
log FDP in Figure 18. If the binding is represented by the

successive equilibria

 

 

E [FDP]
E + FDP -——> EFDP K = Ll__.
F I I [EFDP1]
____s EFDPilLFDP]
[EFDPn_1][FDP]
EFDPn_1 + FDP v—zf‘. EFDPn Kn =

[EFDPn]
and if KI>KII>Kn, then

(I.

or

n(pKav - pEFDPJ) = 108 ga- (2)

where E is enzyme, and a, defined in eq 1, represents the
fraction of enzyme-FDP complex. The theoretical curve in
Figure 18 was calculated assuming n = 2 and the geometrical
average dissociation constant, KD = 63 0M. For similar
treatments of data, see Suelter 22 21° (1966) and references
therein.

If the variation in extent of the first reaction with
increasing FDP concentrations reflects an equilibrium of
enzyme with FDP. and if the fraction of enzyme reacting with
FDP is equal to a, the remaining unreacted enzyme would become
inactivated at the rate observed in the absence of FDP

(defined ask1 and obtained from Figure 16 at zero concentration).

72

.53 no H mm 02m .N n m.e:HESmmm mmpmasoamo ma msfia cfifiom 0:8 .000 000

.MM .0 0o 2H pxmp m£p SH posawmp .0 mm coppoas mam meme 0:9

0H opzmam EH mpdm seapm>apodzH Song ommsfix mumbsaam
endow 000 mam mo 00 pm rm psmhmaa< man we :oHmeHanpoQ .ma enabflm

73

023 $0.: 03
0N KN ON 0..

 

_ _ a _ _ _ _

 

 

NO

v.0

0.0

md

0..

74
Based on this assumption, theoretica1.gT's (slow step) were

calculated for each.FDP concentration.with the use of
e93? = aefig3 + (1 - 0L)8'5151 (3)

where 5T represents the sum of the rates of slow inactivation,
g1 +.33- a is the theoretical fraction of enzyme-FDP complex
derived from the solid line in.Figure 18, i - a is the frac-
tion of free enzyme, 53 is the rate constant of inactivation

for the enzyme-FDP complex, and 5 represents the rate con-

1
stant of inactivation of free enzyme. The theoretical inac—
tivation curves obtained in this manner are plotted as solid
lines in Figure 19. along with the eXperimental points for
each FDP concentration as obtained from Figure 16. The
theoretical intercepts for 0.756 and 1.26 mM FDP (Figure 19)
reflect 98.2 and 99.7% saturation of enzyme with.FDP and

thus are not distinguishable in the experimental data.

The rate constants for the fast inactivation, 32, were
treated by the half-life method (Frost and Pearson, 1961) to
obtain the order of the reaction.with reSpect of FDP. For
any rate expression of the type dg/dt =.£92 - §}2, the half-

life may be defined for all values of‘n 35.2% = £t2m§)[§5'1

where glis some function of‘n and g, and a is defined as the
initial concentration of the reactant. Placing this equation

in logarithmic form yields

log 2% = log _I_‘_ - (£1 - 1) log a (1+)

75

Figure 19. Comparison of EXperimental Data with Theoretical
Determinations of the Zero Time Intercept and
Calculated Rate Constants
Points are taken from the final half of Figure 16.
Intercepts at zero time were calculated using eXperimental
FDP concentrations, and the theoretical curve in.Figure 18.

Solid lines were drawn using calculated rate constants as

described in the text using eq 3.

/X(;'lv1fy

[)cyul 1‘ IC

(‘
‘1

 

 

 

 

200 ‘t 3:: *-*—+* +-+-+-*—+-+_:L
4 u u u
5
\OO 6
80
6O 7
4O

2

‘6

4

.9.

:t: 20
0.

(D
Q.

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77
A log—log plot of 3% 23. 2 should yield a straight line with

slepe (1 - E). Figure 20 is a log-log plot of the té's
obtained from the experimental 32's XE! FDP concentration
from which a value for g = 1.4 was calculated.

iii. Effect of protein concentration on the inacti-
vation

Inactivation was examined as a function of five dif-
ferent protein concentrations (Figure 21). each in the
presence of 1.26 mm FDP. Again inactivation was biphasic
except at very low protein concentrations, where a single-
step inactivation was obtained to the extent that the inacti-
vation.cou1d be followed. Log-log plots of 2%.23- protein
concentration.are shown in.Figure 22a for 52's (fast rate)
and in.Figure 22b for g3's (slow rate). It should be noted
at this point that 1.0 mg/ml of bovine serum albumin in the
0° reaction vessel containing PK at 0.20 mg/ml and FDP at
1.26 m! had no effect on.either 32 or 33.

iv. Sedimentation coefficient of inactivated enzyme

The effect of FDP-enhanced inactivation on the sedi-
mentation coefficient of yeast PK was also examined at 0.8
mg/ml after incubation in the presence or absence of 1.26 mM
FDP at 3.60. Aliquots of protein to be assayed were removed
from the incubation mixtures before loading the centrifuge
cell and maintained at 3.60. Activity remaining was deter-
mined at the time that the first picture of the sedimenting
species was taken. Enzyme minus FDP (95% activity) sedimented
as a single peak (8.6 S). In the presence of FDP, with 30%

78

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79

000 00m

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09 om om on

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Figure

80

21. Effect of Protein Concentration on the FDP-
Enhanced Inactivation of Yeast Pyruvate Kinase
at 00 in 0.1 N Tris-ECI, pH 7.5

FDP concentration in all cases was 1.26 x 10-3 fl.

/\(_;flvofiy

‘)(_’(;0 70¢:

‘4.)

0.5

 

 

81

 

 

 

 

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I00
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33:2 2:0QO

200 250

I50
Time (min)

IOO

5O

82

Figure 22. fieterwination of the Order with HeSpect to
Protein of the FDP-Enhanced Inactivation of
Yeast Pyruvate Kinase at 0°
Both lines are least-squares plots. Rate constants
were determined from the data in Figure 21, as described
in Figure 17. The contribution to the rate of inactivation
by 33 at 0.104 mg/ml was assumed to be negligible, and the

52 in part a at this concentration was assumed to be the

initial rate of inactivation.

83

 

 

 

 

 

 

 

 

3*- 0 k2 /0
5.. e .1.
E e/
V / Slope = 0.53
n=0.47
N
2
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’5 80-
35, 60.
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Protein (mg/ml)

 

84
activity remaining, two peaks could be observed with.§
values of 3 and 8 S in a ratio of about 80:20. With less
than 0.1% activity remaining, in the presence or absence
of FDP, a single peak was observed with anng value of 1.7 S.
2. Inactivation at 23°

1. Dependence of the inactivation on FDP concentration

Although yeast PK is considerably more stable at 23°
than at 0° in the absence of FDP (half-lives differ by nearly
an.order of magnitude) the addition of FDP to the enzyme in
saturating amounts induces an essentially identical lack of
stability at the two temperatures. Data obtained at 23°
showing the effect of increasing FDP concentration from 0 to
2.68 mm on the stability of PK at 0.25 mg/ml are presented
in Figure 23.

ii. Treatment of data from Figure 23

The curves obtained from the semi-logarithmic plot of
specific activity ZE- time are again biphasic as was seen at
0°. Under this assumption, the data shown in Figure 23 were
also treated as in Figure 17. Extrapolation of the data
from the slow step (53) to zero time yields essentially
identical intercepts of Specific activity 110.? i 6.5 units/
mg representing 52% of the initial activity. This is in
contrast to the results obtained at 0° where the intercepts
varied as a function of the FDP concentration. As will be
shown, increasing the FDP concentration above 2.68 mM has no
further effect on the fast step (32), suggesting that this

concentration of FDP was saturating. The 32's obtained from

85

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86

 

 

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80 I20 I60 200 240 280 320 360 400
Time (minutes)

40

87
Figure 23 were treated according to equation 5,
52(x FDP) '.51

a = (5)
£2<2.68 mm FDP) -.Ei

 

where 3 represents the observed rate of inactivation in

l
the absence of FDP (Figure 23); a values were plotted ZE-
log FDP in.Figure 24 as described in the section for 0°
inactivation. The theoretical line in Figure 24 was calcu-
lated assuming 3 = 2 and the geometrical average KD = 0.66 mM.

The rate constants for the slow step (53) were plotted
in.Figure 25 as 1(33 zg. l/FDBE with g = 2.1 giving a
straight line which best fit the data. The KD obtained for
the slow step was 0.52 mM. with a maximum 33 = 1.9 x 10"2

minf1. The maximal.rates of inactivation in the presence

and absence of FDP at 0° and 23° are summarized in Table VI.

TABLE VI

Rates of Inactivation.of Yeastijruvate Kinasea

 

 

Temperature Rate Constants (min'1 I 103)

 

510m) gum) 530mm

 

0° 1.81 102b 10.6
23° 0.235 63.h° 19°

 

QRate constants were determined in 0.1 M Tris-H01, pH 7.5.
at 0.5 mg/ml (0°), and 0.25 mg/ml (23°).

bSaturating FDP = 1.26 mM.

°Saturating FDP 2.68 mM.

88

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89

 

 

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92

iii. Effect of protein concentration on the inacti-
vation

Inactivation at 23° was studied at six different
protein concentrations, in addition to 0.25 mg/ml used in
Figure 23, each in the presence of 2.68 mM FDP. The data
are presented in Figure 26. Again, inactivation was biphasic,
and the extrapolation to zero time of the slow step (53)
gave values which, within eXperimental error, were indepen-
dent of protein concentration and identical to the values
obtained in Figure 23. As at 0° both the fast-step and the
slow-step rate constants increased as the protein concentra-
tion decreased. The data were plotted according to the
half-life method described earlier as log 2% 22! log protein
in Figure 27.

iv. Effect of ionic strength on the inactivation

During efforts to determine the maximal rate of inac-
tivation as a function of FDP concentration (Figure 23), a
decrease in the rate of the slow step (k3) was observed at
concentrations of FDP above 5.36 mM. Increasing concentra-
tions of FDP up to 97.2 mM are presented in.Figure 28.
Identical rates of inactivation were observed at 2.68 mM
(Figure 23) and 5.36 mM (Figure 28) FDP. consistent with
the argument that FDP saturation was achieved with reSpect
to inactivation.before stabilization began. Although the
rate of the second, slow step, is markedly decreased by
high FDP concentrations, no effect on the first step is

observed. (Values for 52 at the high FDP concentrations

93

Figure 26. Effect of Protein Concentration on the FD?-
Enhanced Inactivation of Yeast Pyruvate Kinase
at 23° in 0.1 M Tris-HCl, pH 7.5

FDP concentration in all cases was 2.68 mM.

Specific Activity

94

 

200

IOO
80

60

b
O

N
0

m5

0)

 

 

 

 

 

_Protein (rlglml) D \

1 0.025 ‘ \ e

L o 0.05 ° “
n 0.09 \
e O.l5 A D

A 0.40 \

i. v 0.60 °\ e—
l I I I I I o i

 

IS 30 45 6O 75 9O |O5 IZO
Time (min)

95

Figure 27. Determination of the Order with Respect to
Protein of the FDP-Enhanced Inactivation of
Yeast Pyruvate Kinase at 230

Both lines are least-squares plots. Rate constants

were determined

Fivure 17.

from the data in Figure 26 as described in

(min)

I’)

(filth)

'1/2

+-
onSfZC-S

.ibej K

iv2 (min)

%

 

20

 

 

 

8070
60—

 

k2
.
C
[J I l
I [I I
"3

 

 

Protein (mg/ml)

 

 

97

Figure 28. Stabilizing Effect on.§ of FDP Concentrations
Above 5.36 mm for Yeast Pyruvate Kinase at 230

Enzyme was incubated in 0.1 N TriS°HCl, pH 7.5, at

0.25 mg/ml.

Specific Activity

200

98

 

 

X
Vlv

I60 200
Time (min)

I

l
240

 

V
\

 

l l
280 320

99
are incorporated in Figure 24.)

Since high concentrations of FDP, with a net charge
near 4 at pH 7.5 (Dawson 22 21., 1969) markedly increases
the ionic strength of the solution over the contribution of
the buffer, and since it had been shown that the conforma-
tional change induced by FDP as measured by fluorescence of
PK is inhibited by increasing the ionic strength of the
solution, the possibility that a similar ionic strength
effect, arising from the highly charged FDP molecule itself,
was considered as a potential source of the apparent stabil-
ization. The addition of 0.23 M KCl or (CH3)uNCI in the
presence of saturating (2.68 mM) FDP markedly stabilizes
the enzyme (Figure 29) , although K“ apparently has a much
greater effect. Included in.the Figure are data for the
inactivation at 26.8 mM FDP in the absence of added cation,
a concentration which represents the same ionic strength
as 0.23 M monovalent salt plus 2.68 mM FDP.

In an effort to determine whether the effect of
ionic strength was mediated through the rate of inactiva-
tion, or through the binding constant for FDP, the effect
of increasing FDP concentrations at constant ionic strength
was measured (Figure 30). Ionic strength was maintained
with (CH3)4NCl at 0.934 p, the value at 97.2 mM FDP (upper-
most curve, Figure 28). Rates for both the fast step (32)
and the slow step (33) at 53.6 mM FDP (Figure 30) are
identical as those obtained in Figure 28 at 97.2 mM FDP.

100

Figure 29. Effect of Ionovalent Cations on the FDP-Enhanced
Inactivation of Yeast Pyruvate Kinase at 239

Enzyme was incubated in 0.1 h Tris'HCI, pH 7.5, at
0.25 mg/ml. 0.23 M FCl or (CH3)hNCl in the presence of
2.68 mN FDP represents the same ionic strength as 26.8 mF

F QIT. .

Specific Activity

101

 

200
V V
v v v v
V V
'00 V V V
80
60 0
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40 \o e
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\ ' .

 

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: 0
v 2.68 0
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o 2.68 + 0.23 M (CH3)4NCI
8" v, v 2.68+0.23 M KCI
6 — \'\
V
I l I I V 1 J I J

 

 

4O 80 IZO |60 200 240 280 320
Time (min)

102

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MIAHOV Oiiioads

104

v. Sedimentation coefficient of inactivated enzyme

To examine the effect of FDP-enhanced inactivation on
the molecular weight of the enzyme, the following sedimenta—
tion eXperiments were performed. Enzyme was chromatographed
on a Sephadex column equilibrated with 0.1 M Tris-HCl, pH
7.5. and 0.5 M (CH3)uNCl, and then allowed to stand for
three hours at 23°. The enzyme solution was next divided
in two parts. FDP. to give a final concentration of 45.2
mM was added to the first part, maintaining protein at 5
mg/ml. The second part was adjusted to 5 mg/ml, while
maintaining the ionic strength with (CH3)uNC1 equal to that
of the first part. Aliquots of the two protein samples
were introduced into single sector capillary-type synthetic
boundary cells and mounted in the centrifuge, all at 23°.
After about 30 min, the centrifuge was started, and at the
time of the first picture, the remaining portions of the
enzyme samples were assayed. The sample in the absence of
FDP, with 98% of the original activity remaining, contained
a major component (85%) with an 320,w = 8.5 S, and minor
components (10% and 5%) with sedimentation coefficients of
12.6 S and 8.5 S. reSpectively. 0n the other hand, the
sample in the presence of FDP. with 63% of the original
activity remaining, contained two species with 320," values
of 8.1 S (ZS-30%) and 4.0 8 (70-75%).

To determine whether the 4 8 Species could be
stabilized or possibly reversed to the 8 S species by

removal of the FDP, enzyme was prepared as in the previous

105

eXperiment, and activity was measured as a function of time
after addition of FDP. When activity reached a level of
110 units/mg, an aliquot was removed and chromatographed on
a Sephadex column into the Tris buffer containing 0.5 M
(CH3)4NCl to remove the FDP. Enzyme in the absence of FDP
was similarly treated. Fractions with the highest protein
concentration (2 mg/ml) were immediately introduced into
the centrifuge cells, and sedimentation coefficients were
determined. Enzyme not treated with.FDP again contained
species '1th.§20,w values of 8.3 S (90%) and 12.9 S (10%).
On the other hand, enzyme treated with.FIP contained Species
with 520,w values of 4.2 S (50%) and 2.3 S (50%). Specific
activity at the time of the first picture was only 40
units/mg, and continued to decrease linearly.

vi. Attempted reversal of the FDP-enhanced inacti-
vation of yeast pyruvate kinase

Several attempts were made to reverse the inactivation
produced in the presence of FDP. These included (NH4)2804
precipitation of the enzyme at various stages of inactiva-
tion; rewarming the enzyme solution in the presence of

glycerol, Mg2+, an+

, and combinations of the substrates
and other effectors of yeast PK. All of these attempts were
unsuccessful.

In addition, aliquots of enzyme at various stages of
inactivation at 23° in the Tris buffer, were diluted into

solutions whose composition was known (M. Tobes and C. H.

Suelter, unpublished observation) to yield about 70%

106
reactivation of yeast PK which had previously been dissoci-
ated to subunits with 6 M GuHCl containing 0.15 M 2-mercapto-
ethanol. .Although the diluted aliquots were stable to
further inactivation for at least 12 hr, in no case was any

activity recovered.

DISCUSSION

The primary goal of this research was the elucidation
of the relationship of structural and conformational transi-
tions of yeast pyruvate kinase to the interaction of the
enzyme with substrates and effectors as first observed by
the kinetic studies described by Hunsley and Suelter (1969b).
Fundamental for an analysis of such changes is a character—
ization of the molecular weight of the enzyme and determina-
tion of the size and number of subunits associated therewith.

From sedimentation and diffusion data extrapolated to
zero protein concentration.under two different solvent con-
ditions (Figures 3 and 4; Figures 5 and 6). a weight—average
molecular weight near 167,000 for native yeast pyruvate
kinase was calculated using the Svedberg equation. A
molecular weight near 165,000 was also determined under a
third solvent condition using the high-Speed equilibrium
technique (Figure 7) developed by thantis (1964). Since the
molecular weight of the subunits 18 near 42,000 as determined
for the dissociated enzyme in 6 M GuHCl containing 0.15 M
2-mercaptoethanol. and after maleylation, it is concluded
that yeast PK. like muscle PK (Steinmetz and Deal, 1966), is
composed of four subunits. That these subunits are very

similar in size is consistent with the following observations:

107

108
(1) maleylated enzyme sediments as a single, nearly sym-
metrical peak; (2) the M: of 42,200 from sedimentation and
diffusion data for the maleylated enzyme (Figures 10 and 11)
is in close agreement with MS of 41.400 obtained from sedi-
mentation equilibrium data in GuHCl (Figure 9); (3) the
homogeneous molecular weight distribution for the dissoci-
ated protein throughout the centrifuge cell (Figure 8); and
(4) a single band of molecular weight near 42.000 obtained
by Rose (1969) after electrophoresis of the enzyme in sodium
dodecyl sulfate on sodium dodecyl sulfate-polyacrylamide
gels (Shapiro.g§'§;.. 1967). No evidence is available
regarding the chemical identity of the subunits in this
preparation of pyruvate kinase.

The plots of the molecular weight (Mn) zg. protein
concentration in fringes (Figure 7) shows the presence of
lower molecular weight components at low protein concentra-
tions, indicative of a protein concentration dependent dis-
sociation. This is consistent with the demonstration that
yeast PK exhibits an instability. inversely proportional to
protein concentration. which involves a protein dissociation
to lower molecular weight species, as will be discussed
later. The pattern of}!n as a function of protein concen-
tration (Figure 7) obtained by the meniscus depletion tech-
nique (thantis, 1964) provides, according to Harris 32 al..
(1969), an adequate method for distinguishing between a
protein.participating in a rapid chemical equilibrium. and
a mixture of nonequilibrating or slowly equilibrating Species.

109
A protein in chemical equilibrium should have molecular
weight moments which are a function only of concentration
throughout the cell. Hence, graphs of molecular weight XE.
concentration should superimpose. 0n the other hand, a
heterogeneous system not in chemical equilibrium. or one
participating in a very slow equilibrium should have a
distribution of mass which depends only on distance from
the center of rotation.(Harris gt'al.. 1969); The data
presented in Figure 7 are consistent with the latter situ-
ation. Yet the enzyme at the higher concentration appears
essentially homogeneous with a molecular weight approaching
165,000 in good agreement with the molecular weights calcu-
lated from the Svedberg equation. '

The unusual positive slope of the protein concentra-
tion dependence of 220’" (Figure 11) is an apparent anomaly
which merits further discussion. It can be rationalized on
the basis of extreme charge effects. Spreading of the
boundary in synthetic boundary SXperiments would be eXpected
to be accelerated at higher protein concentrations by the
effect of high concentration of charges. Each maleyl group
bound to the protein changes the net charge by two for each
e—amino group reacted, and by one for each hydroxyl group
reacted.

The data in Figure 6 suggest an unusual effect of
time on.220.w. The sedimentation data in Section I of the
Results indicate that the gross physical structure changes
radically during incubation at 23° in 0.1 M TrisoHCl, pH 7.5,

110
after removal of ammonium sulfate by 0-25 Sephadex. Before
incubation at least three sedimenting Species are observed,
two of which sediment at a rate suggestive of high molecular
weight aggregates of the "native" enzyme. After three hours
of incubation. only one peak is observed. Incubation up to
18 hr leads to no further change in the sedimentation pattern.
However, the results in.Figure 6 indicate that the diffusion
coefficient changes drastically on further incubation. A
plausible conclusion would be that the protein exists as a
diverse mixture of extremely aggregated Species, no one of
which is present in sufficient amount to be observed in the
sedimentation pattern. Apparently these do not affect s20.“
but do affect 1220.". Whether storage of the enzyme as a
suSpension in ammonium sulfate is the cause of the aggrega-
tion is presently unknown.

Considering the wide diversity of solvent conditions
under which the molecular weight studies were performed, it
seems reasonable to conclude that the homotropic and hetero-
tropic substrate and effector interactions observed kinetically
for yeast PK.are mediated directly through conformational
transitions of the enzyme as opposed to subunit association-
disscoiation equilibria of the protein (Frieden, 1967;
Benesch gt 3;” 1966).

The quenching of the fluorescence of the enzyme by
wt and Mg2+ suggests that. like the rabbit muscle PK (Suelter,
1967), yeast PK undergoes a small conformational transition

as a result of interaction with the activating cations.

111

And, as with the rabbit muscle enzyme, the effect is Specific
for KT as opposed to (CH3)4N0. However, the data in Table
III suggest that a more profound effect is produced by FTP
than by the cations taken either separately or together.
The sedimentation data in Table II. while not conclusive
when taken alone. are consistent with this fluorescence
data. That is, a change in sedimentation coefficient sugges-
tive of a conformational transition is observed only under
those conditions which promote a large fluorescence change.
This conformational change, reflected in the changes in
fluorescence and in sedimentation coefficients after addi-
tion of FDP. is analogous to the transition to a kinetically
activated conformer, also induced by F113, and characterized
by a lower Km for PEP.

The apparent antagonistic effect of K+ on the binding
of FDP (Figure 13) is totally unexpected in light of the
fact that this same cation is required, at the concentrations
used in the fluorescence studies, for catalytic activity both
in the presence and in the absence of FDP. The effect appears
to be due to both the increase in ionic strength, since
(CH3)4N“' (Figure 14) yields the same increasing trend in
apparent KD's for FDP. although it does not function
kinetically, and a Specific KI effect, since the extent of
the increase in apparent KD for FTP produced by (CH3)4N+ is
not as great as by K+. A Specific K+ effect is also supported
by the unequal stabilizing effects of the two cations during
the FDP-enhanced inactivation at 23° (Figure 29). The

112
antagonistic effect of ionic strength on the binding of FDP
is also observed in the catalytic assay using sub Km concen-
trations of AIP. PEP. PIP. and 10* (0.5. 0.087, 0.027, and
10.0 mM. respectively). Increasing the KT from 0.01 M to
0.23 M (in a single assay) decreases the observed activity
by an order of magnitude, the rate of decrease having a
half-life on the order of seconds. Addition of 0.23 M
(CH3) “14+ has a similar effect, although the decrease in
activity is only about half as great as in the case with
KT. Similarly. the observation (Table III) that KT quenches,
to a limited extent. the fluorescence of yeast PK, whereas
(033mm does not, supports a specific cation effect in
addition to the effect of ionic strength.

The most marked influence on the binding of FDP to
yeast PK is exerted by PEP in conjunction with K+ (Table
IV), an effect which might be expected, Since kinetically,
the Km's and 2H'3 for PEP are markedly affected by FDP
(Hunsley and Suelter. 1969b). The affinity of the enzyme
for FDP in.the presence of PEP and KT is increased by
almost two orders of magnitude over the affinity at the
analogous K+ concentration in the absence of PEP. 0n the
other hand. PEP alone, and PEP in the presence of 0.23 M
(CH3)hN+ do not decrease the KB for FDP, suggestive of a
Specific monovalent cation-substrate-enzyme complex involved
in.the FDP-induced conversion.of the enzyme to its activated
conformer. The implication that K+ might bind at or very
near the PEP binding site on the enzyme is consistent with

113
the nuclear magnetic resonance data of Kayne and Reuben
(1970) using TI+ and rabbit muscle PK. Suelter (1970) has
recently suggested that K+-activated enzymes may involve the
monovalent cation in a ternary enzyme-substrate complex pg:
33, implying binding at the active site.

The observation that ADP in the presence of cations
also affects the dissociation constant for FDP with yeast PK
is surprising. Kinetically, FDP has little or no effect on
the Km for ADP and no heterotropic or homotropic cooperativ-
ity has been Shown involving ADP. with the Hill SIOpe equal
to unity under all conditions (Hunsley and Suelter, 1969b).
The addition of 5 mM ADP in the presence of 0.23 M K+ or
(CH3)uN0, however, reduces the KB for FDP. as determined by
the fluorescence titrations, by an order of magnitude.
Further, the Hill Slopes near 2 in the absence of ADP were
reduced to unity in its presence, suggesting a significant
role for ADP in the determination of conformation of the
enzyme, a conclusion not apparent from the kinetic data.
Unlike the results obtained for PEP. the enzyme-ADP inter-
action which is reflected in the KD for FDP is not mediated
by monovalent cations, and occurs in the absence of both K+
and (CH3)4N+. Further, the effect of ADP appears to be the
dominant interaction. sufficiently strong to reverse the
antagonism between monovalent cation and FDP.

The substantial increase in KD for FDP as the temper—
ature is raised from 0 to 30° (as determined from fluores-

cence changes in the presence of Mg2+) agrees with the

114
similar increase in KD monitored by the FDP-enhanced inacti-
vation of the enzyme in the absence of Mg2+ (summarized, in
Table VII). These observations are consistent with a con-

former of yeast PK favored by both low temperatures and FDP.

TABLE VII

Temperature Effects of K 's for FDP and
Yeast PK in 0.1 M Tris- Cl, pH 7.5 by
Fluorescence Changes and Inactivation Data

 

 

 

 

 

Method Temperature KD (mM)

(1) Fluorescence8 0 0.123
23 0.487

30 0.912

(2) Inactivation 0 0.063
23 0.660

 

8In the presence of 0.025 M MgC12.

Although an identical Vmax is obtained for yeast PK
in the presence or absence of FDP, and both conditions
require K+ (Hunsley and Suelter, 1969b), the data in Table
III indicate that 0.23 n 10‘. even in the presence of Mg2+
and PEP. does not produce the same fluorescence change in
PK as is obtained with FDP. If the fluorescence properties
of the enzyme reflect its conformation. and if the Spectrum
in the presence of FDP reflects an activated conformer. one
of two alternatives could be considered. 0n the one hand,

the fluorescence of the enzyme in the presence of K+, Mg2+.

 

115
and‘PEP would reflect a conformation of the enzyme which is
intermediate on the path to the kinetically active conforma-
tion. In this case ADP is required to complete the confor-
mational transition. Such a possibility cannot be excluded
on the basis of kinetic data, Since ADP apparently alters
the structure of the enzyme in such a manner as to facilitate
FIP binding. as shown in Table IV.

On the other hand, if ALP does not provide the driving
force to complete the conformational transition, then it
would seem necessary to argue the existence of two different
active conformers controlled by FDP and KT. These would be
distinguished by their fluorescent properties, and kinet-
ically. by differences in Km's for PEP. This second alterna-
tive is supported by the antagonistic effect of K+ on the
binding of FDP. and further suggests a dual role for K+:

(1) as a required cation for catalysis in the presence and
absence of FDP and (2) in producing an apparently unique
active conformer in the absence of FDP. Such a dual role

is also consistent with the observation (Hunsley and Suelter,
1969b) that Na+ can substitute for K+ only in the presence

of FDP.

0n the basis of the fluorescence data, the suggestion
that FDP promotes a major conformational transition of yeast
PK seems well-founded. The relationship between this struc-
tural change, observed in the presence of Mg2+. and the
inactivation enhanced by FDP in the absence of Mg2+, depends

to a considerable extent on whether the conformational

116
changes involved in each case are identical or at least
similar, and whether FDP in each case binds at the same site
on the enzyme.

It seems unlikely that two distinct sites exist on
the enzyme for FDP. one site promoting an activation, and
the second site promoting an inactivation. Protection by
"82+.l2l1312 would require extensive compartmentalization
and/or regulation of the uptake of this cation for the
inactivating role of FDP to be metabolically operative.

The x+ content of the cell should also be sufficient to
protect the enzyme from inactivation. In addition, the FDP
activation as a regulatory mechanism for glycolysis makes
the second site alternative highly improbable. If the
effects on the kinetic properties of yeast PK and on the
stability of the enzyme result from ligand binding at the
same site, then binding of the divalent cation in conjunc-
tion with FDP could promote a conformational change leading
to an activated enzyme form. Without a divalent cation,
the enzyme would reSpond "incorrectly" or incompletely to
the bound FIP, and would dissociate.

Alternatively. Mg2+ may function as a stabilizing
factor. Binding of FDP in the presence or absence of the
metal may produce identical structural effects on.PK. but
the divalent cation is required to stabilize the newly
induced conformation. A combination of these two alterna-
tives seems most likely: 3.3.. binding of FDP might induce

only a partial conformational change to a highly unstable

117
form of the enzyme, which, in the presence of cation, would
be completed to the fully active, stabilized conformer. The

fact that FDP in the absence of Mg2+

produces a very similar
though slightly smaller quenching of the fluorescence of PK,
as compared to that obtained in the presence of the metal.
supports this contention. In addition, the analogous effects
of temperature and ionic strength (including the apparent
Specific and general monovalent cation effects) on the
fluorescence change produced by FDP and the instability of
the enzyme induced by the ligand are consistent with the
implication of identical binding Sites and similar confor-
mational changes in both processes. With this as an assump-
tion. an analysis of the inactivation mechanism may reveal
information concerning the structural changes involved in
the ligand binding during the catalytic process.

The sedimentation data for yeast pyruvate kinase in
the presence and absence of FDP at 23°, and the inverse
dependence of both steps of the inactivation on protein con-
centration (Figure 27) are consistent with a mechanism of
inactivation involving two consecutive dissociations of the
native tetramer to inactive monomers. If the 2.1 8 Species
represents the partially unfolded monomer of molecular
weight 42,000 [a folded globular protein of this molecular
weight would be expected to have an.§ of about 2.4 S
(Holleman. 1966)], the 4 S Species suggests the existence
of a partially unfolded dimer as an intermediate in the
inactivation (eq 6a) .

118
8.5 S —————+> 4 S-———-—€> 2.1 S (6a)

Similarly, the dependence of the rapid (52) and Slow
(53) steps of the inactivation at 0° on protein concentra-
tion (Figure 22) and the sedimentation pattern of the enzyme
at various stages of the inactivation, are consistent with a

protein dissociation into subunits (eq 6b). The 3 S Species
8.6 s-——-—€> 3 S —-- > 1.7 8 (6b)

represents either an extensively unfolded dimer, or a mix-
ture of dimer and monomer resulting in an apparent g = 3 S,
and the 1.7 8 Species correSpondS to an unfolded monomer
produced by the slow inactivation, k3. The biphasic inacti-
vation curve obtained at 0° in the presence or absence of
F11D (Figure 1 and Figure 16) and the appearance of the 1.7 S
Species after nearly complete cold inactivation, also in the
presence or absence of FDP. suggest that similar mechanisms
of inactivation are operative in both cases.

Although similar two-step protein dissociations appear
Operative at both 0° and 23° in the FDP-enhanced inactivation.
a comparison (Figure 16 13. Figure 23) of the characteristics
of the effects of FDP on the course of the inactivation at
each temperature is difficult to reconcile without the exis-
tence of alternate pathways from tetramer to monomer. For
this reason the overall inactivation scheme as presented in
Figure 31 is proposed.

That the binding of FDP to yeast PK in the absence of

119

0mm one 0 pm mommax opm>5pmm
pmmow mo Coapm>fipomcH poozm£CMImmm mo Emficmsomz pmmoaoem

.Hm meaeaa

 

 

 

 

 

121
Mg2+ promotes an alteration in quaternary structure is, of
course, a conclusion reached directly from the observation
that the ligand promotes a rapid inactivation of the enzyme.
In the presence of saturating FDP at 0° the rapid rate of
inactivation. 32 (Figure 22a), is dependent on protein con-
centration. suggesting that protein dissociation is then
the rate-limiting step. The data also suggest (Figure 18
and eq 2) that FDP enhances the inactivation by promoting
a shift in the equilibrium between the two tetrameric con-
formers in Figure 31, and that a minimum of two moles of
FDP bind per mole of enzyme in shifting this equilibrium.
Wicker gt a}. (1969) have Shown that two moles of FDP bind
per mole of native enzyme consistent with this suggestion.
The interaction constant of 2 for FTP (Figure 18) in defin-
ing the equilibrium between tetramer and 0° dimer following
the rapid inactivation is also consistent with the above
model. The kinetic order of 1.4 for interaction of FDP at
0° (Figure 20) suggests a complex series of steps involved
before establishment of the inactivation equilibrium. How-
ever, the interaction constant of 2 in Figure 18 suggests
that the concentration of the intermediate complexes with
FDP which might occur during the inactivation are insignifi-
cant after the equilibrium is established. Such a mechanism
is analogous to the dissociation of a diprotic acid in which
two protons dissociate with no detectable amount of an
intermediate singly dissociated form in the equilibrium

mixture (Suelter g§|§l., 1966, and references therein).

122

While the data at 0° are consistent with the estab—
lishment of an inactivation equilibrium between tetramer
and dimer as reflected in the dependence of the rapid step
of inactivation on both FDP (Figures 16 and 18) and protein
concentration (Figure 21), the extent of this step at 23°,
proceeding to 50% inactivation, is independent of either of
these variables (Figures 23 and 26). The correSponding
changes in sedimentation coefficient suggest that this activ-
ity may be attributed to the 4 S Species, a conclusion con-
sistent with the lack of any observable 8 8 Species obtained
after removal.of FEP by treatment with Sephadex, even though
approximately 25% of the initial activity remained. Although
the dimer obtained at 0° could be partially active, attribut-
ing a Specific activity of 50% of the native enzyme to the
0° dimer would not be consistent with the data, and Suggests
that the dimers obtained at 0° and at 23° are different.

The argument for two different dimers after the first
inactivation step at the two temperatures is also consistent
with the divergent nature of their formation. The extent of
dimer formation at 23° is independent of FDP and protein
concentration (Figures 23 and 26), that is, the first step
of inactivation at 23° appears essentially irreversible. At
0° the extent of dimer formation is dependent on botthDP
and protein concentration (Figures 16 and 21). The depen—
dence of the rate of the second step of inactivation on FDP
concentration.(Figure 25) suggests a conformational transi-

tion of the dimer. Since the removal of FDP after completion

123

of the first step of inactivation at 23° led to an inacti-
vation of the dimer, at 0.5 u. much faster than the rate
observed at the comparable ionic strength maintained with
FDP (53.6 mM FDP, Figure 28), but slower than the maximum
rate at saturating FDP (Figure 23), it would appear that
FDP at high ionic strengths contributes some stability to
the enzyme. Such an effect would suggest a second altera—
tion in the conformation of the 23° dimer, perhaps by a
mechanism consistent with a dissociation of the bound FDP
followed by a rebinding of the ligand.

The unusual effect of high concentrations of FTP on
33 at 230 (Figure 28) is also consistent, however, with an
inhibition by ionic strength of both the binding of FDP to
the dimer as well as the dissociation of the dimer to inac-
tive monomers. The first effect is suggested by the data
in both Figures 29 and 30, and the observation that increased
ionic strength markedly inhibits the binding of FDP as
measured by changes in the enzyme fluorescence. When ionic
strength is maintained at 0.934 u (Figure 30). the apparent
ED for FDP increased from 0.66 mM to approximately 5 mM as
reflected in 52. and from 0.56 mM to about 4 mM as reflected
in‘ka. However, the maximum rate obtained for £2 at saturat-
ing FDP is independent of ionic strength, whereas 53 at
saturating FDP is markedly decreased in the medium of high
ionic strength. The invariance of £2 with ionic strength
(Figure 28) does not contradict the above conclusions, but

rather suggests that the FDP concentration always remained

124

sufficiently high to out weigh the eXpected effect of ionic
strength on the dissociation constant for FDP with enzyme.

If the inactivation from tetramer to monomer, facil-
itated by the presence of FDP. proceeds by alternate path-
ways of cleavage of the tetramer at the two temperatures
examined, as depicted in Figure 31. heterologous interac—
tions between the subunits of yeast pyruvate kinase would p
seem likely. The temperature dependence of inactivation in
the absence of FDP (Figure 1) suggests a break in the stabil-
ity curves between 6 and 0°, reminiscent of similar sharp 9
changes in stability of cold labile enzymes (for example, ’
pyruvate carboxylase, Scrutton and Utter, 1965; 17-3-
hydroxystercid dehydrogenase, Jarabak 33 21., 1966). Pos-
sibly the increased structuring of water, which occurs near
4°, may be influential in creating the sudden instability of
this enzyme in this temperature region.

Low-temperature instability of proteins indicates
that associations between apolar groups are important in
these proteins, since hydrophobic bonding would be expected
to be significantly weakened at low temperatures (Kauzmann,
1959; Scheraga 22 21., 1962). Although little can be said
with certainty concerning the structure of pure water
(Eisenberg and Kauzmann, 1969) thermodynamic analyses of the
interaction of apolar groups with water indicate a large
unfavorable entropy loss as the temperature is lowered,

which may be attributed to a major change in the structuring

125
of water around the apolar group (Kauzmann. 1954) in some
manner analogous to the "iceberg" concept of Frank and
Evans (1945). Brandts (1969) has suggested that the favor-
able free energy change involved in removing an apolar
residue from an aqueous medium is almost entirely entr0pic,
and due to the concomitant "melting" of the "clathrate"
water structure around the residue. Hence, the optimum
temperature for stability of a protein should increase as
the hydrophobicity of the protein increases. This holds
true for chymotrypsin.zg. ribonuclease. von.Hippel and
Waugh (1955) in describing the nature of the low tempera-
ture dissociation of casein, observed that "... casein has
an unusually high number of non-polar residues ..." Hence,
the effects of temperature and glycerol on the stability
of yeast PK implicate water structure, and Specifically
hydrophobic bonding in the associations between the subunits
of the enzyme.

The suggestion that different dimers are obtained
during the FDP enhanced inactivation of yeast PK depending
on the temperature involved, and the conclusion that heter-
ologous subunit interactions exist in the native enzyme,
implies a greater degree of hydrOphobicity in the bonding
between the subunits of one of the dimers. If the dissocia-
tion from tetramer to dimer at 0° favors a Splitting of the
enzyme along hydrophobic bonds, then it would be eXpected
that the dissociation at 23° would lead to dimers with pre-
dominantly the same hydrophobic bonds between the subunits

 

s»

:br.

126
of the dimer. The stabilization of the 23° dimer by high
ionic strength (Figure 30 253 Figure 23), which would be
expected to favor association of apolar groups (von Hippel
and Schleich. 1969), supports the inclusion of a relatively
large number of such hydrophobic bonds between the Subunits
of the 23° dimer. Similarly, the lack of any effect of
high ionic strength on the maximal value obtained for k2
(Figures 23, 28, and 30) might suggest that electrostatic
forces play a predominant role in the association of these
dimers in the native tetramer, as well as between the sub—
units of the 0° dimer.

The analogous effects of temperature and ionic
strength on the binding of FIP to yeast PK observed both
through measurement of fluorescence changes and through the
inactivation phenomenon, as well as the effect of increased
ionic strength on the kinetic properties of the enzyme,
strongly support the argument that the structural changes
observed in yeast PK during its inactivation by FDP reflect
more subtle changes in the conformation of the enzyme,
which yield the kinetic activation. by the same ligand. in
the presence of substrates and divalent cation. A model
(not meant to be unique) for kinetic activation of yeast PK
by FDP consistent with the data obtained from the inactiva-
tion phenomenon, would involve a change. concomitant with
the ligand binding, in the conformation of the individual
subunits of the tetramer to a more compact form, with a

resultant decrease in the area of contact between the sub—

127
units. This decreased contact between the subunits would be
accompanied by exposure of the residues of the polypeptide
chains, which normally interact strongly when the enzyme is
in the nonpactivated state. Decreasing the interaction
between the subunits should result in a reduction in 000p-
erativity of ligand binding to the enzyme. This effect is
obtained kinetically as the Hill slopes for all ligands in
the pyruvate kinase reaction decrease in the presence of
FDP (Hunsley and Suelter, 1969b).

The increase in apparent KD for FDP observed in the
fluorescence titrations as ionic strength is increased, does
not necessarily reflect an inhibition of the actual binding
phenomenon, but rather represents an inhibition of the struc-
tural transition of the enzyme from the noneactivated state
(with low affinity for FDP) to the activated state (with
high affinity for FDP). If the residues which are exposed
during this transition are predominantly hydrophobic in
nature, high ionic strength would be eXpected to inhibit
the transition by FDP as is observed not only in the fluores—
cence measurements, but also during the inactivation
phenomenon, and in the kinetic assays. Similarly, glycerol,
which would be eXpected to disrupt water structures around
hydrophobic residues, and thus eliminate the unfavorable
entropy change associated with the separation of apolar
residues in an aqueous environment, would thus also be
expected to decrease the cooperativity of protein-ligand

interactions, and to decrease the kinetically activating

 

128
effects of FDP. These effects have also been observed with
yeast PK (M. Ruwart and C. H. Suelter, unpublished observa-
tions). Finally. low temperatures, which would favor dis—
ruption of hydrophobic bonding by allowing for the structur-
ing of water around the apolar residues, would be expected
to also favor the conformational transition induced by FDP.
This effect of temperature is reflected in the decrease in
apparent KD'S for FDP as measured both by the fluorescence
changes, and in the inactivation phenomenon as the tempera-
ture is lowered. It might also be expected that low tem-
peratures would activate yeast PK somewhat like FDP activates
the enzyme (see below for this type of temperature effect in
PK isolated from Alaskan king crab).

The delicate interplay of forces which apparently
exists in regulating the conformations of yeast pyruvate
kinase may reflect a more general phenomenon particularly
significant to those enzymes which exhibit regulatory
properties. The exposure of hydrophobic residues, concomi-
tant with structural transitions, would contribute signifi-
cantly to the cooperativity of such transitions by intro-
ducing a significant free energy change as these apolar
groups interact with the surrounding environment. The char-
acteristics of the solvent (such as ionic strength, or the
presence of polyhydroxic compounds or their physiological
counterparts) thus play a major role in maintaining the

subtle balance between conformational states of an enzyme.

129

In conclusion, it is interesting to examine other
preparations of pyruvate kinase with reSpect to the effects
of temperature on their structure and kinetic properties.
Kayne and Suelter (1968) have described conformational tran-
sitions of the rabbit muscle enzyme as observed by ultra-
violet difference Spectra, changes in the sedimentation
coefficient, and changes in optical rotatory diSpersion
parameters. These data indicated structural changes in the
protein as the temperature was lowered. The Km for PEP of
the pyruvate kinase isolated from Trematomus bernacchii
(Somero and Hochachka. 1968) decreases as the temperature
is lowered to 5°. This temperature represents the habitat
temperature of the arctic fish. Finally, the pyruvate
kinase isolated from Alaskan king crab (Somero. 1969) has
been shown to consist of two interconvertible forms, con-
trolled by the temperature. The "cold" form exhibits
hyperbolic kinetics, and has a minimal Km for PEP at 5°.
Because of the unfavorable Km at higher temperatures, this
form only functions below 10°. The "warm" form has a mini-
mal Km for PEP at 12°. and exhibits sigmoid kinetics. Thus,
lowering the temperature induces a conformational change
with effects analogous to addition of FDP to the yeast
pyruvate kinase.

The biological Significance of the interconversion
of PK forms in the Alaskan king crab is that the organism
can function equally as efficiently, at least in this

enzymic reaction, throughout the entire range of habitat

130
temperatures which it will encounter. Its adaptation to
temperature changes is thus rapid and efficient. The
ability of an organism to adapt to temperature variations
is essential for survival. The structural and conforma-
tional transitions described in this Discussion may suggest
the involvement of subtle changes in protein structure or
inter-cellular environment in the mechanism of such bio-
logical phenomena as hibernation and cold hardiness. The
possibility of :radical changes in the primary structure
of enzymes during these phenomena cannot be discounted
either. For example, Roberts (1969) has recently presented
an interesting Speculative review on the role of isozymic
substitutions during cold hardening in plants. Examination
of the enzymes and cellular compositions of organisms during
these phenomena may provide the insight into the exact

mechanisms involved .

 

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