~15"'5‘555555" .I'.‘ 55'/II
55 55II555II. '555 5555555555555 .

I55

55 55. .I' ,. .. ~ . , .
.55.. . .5 .. . .I. , ..
5555555555555 5: 555551“! 55.55555”:I ' 5555555555555”5'55““...555555555 5‘ 5‘
I55

I

5575555 555m; 555‘ I555 55,5555

5. 55555555

ga'5 55555555 55. 5555555555555 5"5'5 ”I'm?” .
' 5555"'5' 55' 5‘5 ‘5"5 2 ~
5‘55 555555 5555555555» 555555555 5."

55"

I 5 . I 5 5 "55‘. 5.55

... 5 55

'I.'
I5 '55555'5
5555 55 5'5'55555
‘" ‘55." III 55'5' "" "I“III‘I 5.555

'5'5. , III III 5"" 555555555“ 555 55555:; ' 5,
55555555 555 55555 55555555 '5'55 '55555 55'5‘ 555555555 5.55. 5 '555, 5.. 555 5555555 55 55.5555
'5' 555555555 '5 55555 ' '55' 55955 55.55. 555555 5.5555 55555 ‘5. 55555 5555555 5555....

55555 ..5 55' 5 H.555 'III ‘-"II ‘N 555555' "'5

I 55. ""5'.5' I55.555 5".555'553'5 ”513.455.5555

55. I. 5.5555 555‘ '
5 55 '55'55" III'" 5 5 I 5 555555I 5 55 555 . 5 55'. 5 HI 5 55"5515'555
"55I ' " "
.‘5 5

5555555 5555 555555

   

  
   

 

'."l’..':'-555 555 "55‘|', .5555 5‘ 5 55555.
" 5555' . :..' , "'55! .7.5~;‘~~';; 5‘5 .';:.; 5.55.5» " ,. ...:N
.5 '5‘ III 5.55 5 ‘5 ... IIII .,,5,,.55,555, ... .-.-
1.. '5)! '.

1"". “€51:
"" '55 5",5‘ , I :5
' "5 555’5 '5Qk .6

      
 
 
 

    

551455.55 : 55 I5,‘ '555'
55.55535" 5555.55 « 5,: 55'

 

 

 
 

555! 5... 5.5 555 5555'

. 5‘5
.' $55 " 5'555555555.5 555,5,..""55" 5555.5‘155"' 5 5
' I 555555 5'5" ' ".""'5'="5"  I 5 "‘5' 55" "5‘5. ..5 '.I.. '
”5'555555555555' """."' 5 " " ' ' ' ' "5 "'" '

.5 . 5,5,55,55,55 .555." ...} 5 ,5.“ '5. It. 5.5. 5 55.. 555-5‘. 5555 555:. 5,553.,
9555555 555555555 .. 55555' 5, 5' ,..'I" '55'155' '5555'5 5 "'5 55. 555555. 5555555 555555 5555 55555555 555555 55555555555555.555’ 354$

555 '55' I
”55" 555.';'5,5' II, 55 5‘5"' 55 .5‘5 .."55 5' 555555555' 55555 555 555'". l"'5""5 "" '555 I555

 

. .. .j-5 I 55‘5
.5.. , . 55“ I.
555.55551'5' ' "' " 5' "'II

I 5 '55': " 'I
"' ‘ .5555 ' - 5 ' 55' l I"' 5
II' 5"." ""' " . I . .5" O . ‘ I 5. 'I 5. " ~ ' '.- 555' -'
5 5 .555 55555' 55: 5I 5. ,5 ' I ' .51555 5555555. . “5.55.5555. . ‘ 5 I .""' .55555W
..l 5 5'." ':55. I I 55 ..‘ .5 5.5.5.1..5...“ n... .I ...... .5...... ‘. 5L1“. ML... .‘............u.I..LI............m.i ._ ..‘."I I ....5. [Int '

m... IlIIIIIIIIIlIII I III2

 

This is to certify that the

thesis entitled

STUDIES ON THE MECHANISM OF TRYPTOPHANASE
CATALYSIS

presented by
DAVID SHERIDAN JUNE

has been accepted towards fulfillment
of the requirements for

PH.D. deg-99in BIOCHEMISTRY

WW

Major professor

Date M

0—7 639

 

OVERDUE FINES ARE 25c PER DAY _
PER ITEM

Return to book dr0p to remove
this checkout from your record.

I

   

 

 

 

~ - §- w*-——,i

 

STUDIES ON THE MECHANISM OF TRYPTOPHANASE CATALYSIS

By

David S. June

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirement5°

for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1979

ABSTRACT
STUDIES ON THE MECHANISM or TRYPTOPHANASE CATALYSIS
By

David S. June

In an attempt to gain insight into the mechanism of
tryptOphanase catalysis and to further elucidate the role of
monovalent cations in the catalytic process, equilibrium and
kinetic studies were performed in the presence and absence
of substrates and inhibitors under a variety of experimental
conditions. Experimentally, this research was divided into
three parts, each dealing with separate but interrelated
characteristics of the enzyme. The results of these investi-
gations were integrated into a simple model of tryptOphanase
catalysis and monovalent cation activation.

The first part of this research deals with the effects
of pH and monovalent cations on the spectral and kinetic
properties of tryptOphanase. The apparent pKa value for the
Spectral interconversion of the 420 nm and 337 nm forms of
the enzyme as a function of pH was determined in saturating
concentrations of NH~*, K’, Rb+, Cs+, and Li+. The apparent
pKa for this SpectrOphotometric titration was found to be
inversely prOportional to the Vmax obtained in the various

cations using S-orthonitrothiOphenyl-L-cysteine (SOPC) as

David 8. June

the substrate. A simple mechanism was proposed to explain
the simultaneous increase in pKa' and decrease in Vmax in
poor cation activators.

Values of Km and Vmax for the degradation of SOPC and
KD for the binding of L-alanine to tryptOphanase were deter-
mined over the range of enzyme stability. The results were
consistent with the interpretation that a zwitterionic amino
acid binds to an enzyme molecule following deprotonation of
a functional group on the protein with an apparent pKa in
the range 8.2 - 8.5. Vmax was independent of pH in both
K+ and Li+ over the range of pH studied.

The second part of this research deals with scanning
stepped flow studies on the kinetics and mechanism of the
pH-dependent interconversion of the spectral forms of
tryptOphanase. These studies, which involved incremental
pH jumps and drops over the range of enzyme stability, demon-
strated that the spectral forms of tryptOphanase interconvert
in a complex fashion on the stOpped flow time scale following
a rapid change in pH. These spectral changes were analyzed
in terms of three distinct phases: 1) an abrupt phase,
which is complete in less than S msec, 2) a fast first order
interconversion of 420 nm and 337 nm absorbance, and 3) a
slow first order process involving growth at 355 nm coupled
to two decays centered at 325 nm and 430 nm in the incre-

mental pH jumps; and decay at 355 nm with concomitant growth

David 8. June

at 430 nm and 290 nm in the case of the incremental pH dr0p
experiments. Major features of the data were interpreted
in terms of a simple model including kinetic constants and
postulated structures.

The final section of this thesis covers scanning stapped
flow studies on the mechanism of quinonoid formation with
tryptOphanase using the competitive inhibitors L-alanine
and L-ethionine. The effects of pH, concentration of inhib-
itor, differing monovalent cations, and substitution of
deuterium at the a-position of alanine on this reaction were
examined. These results were integrated with the findings
of the earlier studies into a simple mechanism of trypto-

phanase catalysis up to and including a-proton abstraction.

DEDICATION

To Jennifer and Meredith

ii

ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to
Dr. Clarence H. Suelter for being a friend as well as
an excellent thesis advisor. Thanks also to Dr. James L.
Dye for his sagacious advice and guidance. Financial
support by the National Institute of Health and the
National Science Foundation is also acknowledged.

I am grateful to Ann Aust, Mark Brody, David Husic,
Gerard Oakley, Mary Pearce, Debra Thompson and Shyun
Long-Yun; some fine peOple who helped make my stay at
MSU more pleasant and productive. My parents, whose
unquestioning support helped me more than they know,
deserve special thanks. And finally, to my wife
Jennifer, thank you for making these four years mean-

ingful.

iii

TABLE OF CONTENTS

List of Tables

List of Figures

List of Abbreviations

Organization

1.

II.

III.

Introduction

A Brief Overview of Pyridoxal-P Catalysis

Literature Survey

References
Effects of pH and Monovalent Cations on the
Spectral Characteristics and Kinetic
Preperties of Tryptophanase

Introduction

Materials and Methods

Results

Discussion

References
Studies on the Kinetics and Mechanism of the
pH-dependent Interconversion of the Spectral
Forms of Tryptophanase

Introduction

Materials and Methods

Results

Interpretation of Experimental Results
and Discussion

References

iv

Page
vi

viii
xi

xii

14

16
16
21
46
53

SS
57
91

107

Table of Contents - continued

Page
IV. Scanning Stapped Flow Studies on the
Mechanism of Quinonoid Formation with
TryptOphanase using Competitive Inhibitors
Introduction 109
Materials and Methods 110
Results 114
Discussion 146
References 153
Appendix A 155
Appendix B 161

Table

II.

II.

III.

II.

LIST OF TABLES
Page
Section I

Activating constants for Monovalent cations 7

Section II

The effect of monovalent cations on the 32
apparent pKa for the interconversion of the
420 nm and 337 nm forms of tryptophanase.

Kinetic parameters for the degradation of 47
SOPC and the formation of the quinonoid
with L-alanine as a function of pH.

Section III
Kinetic parameters for the fast first order 79
interconversion of 420 nm and 337 nm absor-

bances in the pH jump and drOp experiments.

Kinetic parameters for the slow first order 88
process in the incremental pH jump experiments.

The percentages of total enzyme that exist 97
in each of the four forms H EB, BB, H EY and
EY at various pH values.

Section IV
Kinetic parameters for the biphasic growth 123
of quinonoid as a function of ethionine
concentration.

Kinetic parameters for biphasic quinonoid 127
growth at three concentrations of alanine.

vi

List of Tables - continued

Table
III.

IV.

Kinetic parameters for the absorbance
changes at 337 nm, 420 nm and 508 nm
which occur when tryptOphanase is mixed
with ethionine.

Kinetic parameters for the triphasic
growth of quinonoid at pH values 7.2,
8.0 and 8.7.

Kinetic parameters for the biphasic
growth of quinonoid observed when a
tryptOphanase-ethionine complex is
mixed with monovalent cations.

vii

Page
134

138

142

Figure

LIST OF FIGURES

Page
Section II

Effect of various buffers on tryptOphanase 23
activity.

Plots of the absorbance of tryptophanase 25
at 420 nm and 337 nm measured as a
function of pH.

Relationship between Vmax for the 36
degradation of SOPC and pKa' for the
interconversion of the 420 nm and 337 nm

forms of tryptophanase.

The pH dependence of the dissociation 39
constant, KD, for L-alanine.

Variation of the affinity of tryptOphanase 42
for SOPC and the maximal velocity with SOPC.

Section III

Absorbance Spectrum of tryptophanase at low 59
and high pH values and absorbance difference -
wavelength - time surface observed in a pH

drOp experiment.

Abrupt difference Spectra obtained in the 63
incremental pH jump and drOp experiments.

The change in absorbance at 295 nm as a 69
function of pH for the abrupt spectral

changes observed in the incremental pH

jump and drOp experiments.

Spectral changes occurring during the fast 73

first order interconversion of 420 nm and
337 nm absorptions.

viii

List

Figure

10

11

of Figures - continued

The changes in absorbance at 337 nm and
420 nm observed in the incremental pH
jump and drOp experiments as a function
of pH.

Changes in absorbance at 295 nm, 337 nm
and 420 nm as a function of time during
the fast first order conversion of 420 nm
to 337 nm absorbance following a jump in
pH from 7.00 to 9.33.

Spectral changes occurring during the
slow first order process following a
rapid change in pH.

The change in absorbance at 355 nm as

a function of pH observed in the slow

first order process in the incremental
pH jump experiments.

Variation of the apparent first order
rate constant for the fast first order
interconversion of 420 nm and 337 nm
absorptions in the incremental pH jump
and drOp experiments.

Absorption spectra of various ionic
forms of pyridoxal-P. HzPL (0),
HPL (A), PL (+).

Difference Spectrum obtained by subtracting

the HzPL Spectrum in Figure 10 from the HPL
spectrum,

ix

Page

77

81

86

90

96

100

102

List of Figures - continued

Figure

Section IV

Tryptophanase spectra collected as a
function of time after mixing with
ethionine.

TryptOphanase spectra collected as a
function of time after mixing with
alanine.

The biphasic growth in absorbance
at 508 nm after mixing tryptOphanase
with ethionine.

The variation of the apparent first
order rate constants, ,' and k2'

for the fast and slow phases, reSpec-

tively, of the biphasic growth of
quinonoid with et

The changes in the tryptophanase
absorption Spectrum at 337 nm and
420 nm as a function of time after
mixing with ethionine.

The rate and extent of quinonoid
formation as a function of pH.

The rate and extent of quinonoid
formation in the presence of various
monovalent cations.

The effect of deuterium substitution
at the a-position of alanine on the

rate and extent of quinonoid formation

with tryptophanase.

ionine concentration.

Page

116

119

121

126

130

136

141

145

Bicine
EDTA

Epps

Mes

pKa'
pyridoxal-P
SOPC

Tes

LIST OF ABBREVIATIONS

N,N-bis (2-hydroxyethyl) glycine
ethylenediaminetetraacetic acid

N-2-hydroxyethylpiperazine prOpane
sulfonic acid

2 (N-morpholino)ethane sulfonic acid
apparent pKa

pyridoxal-5'-ph05phate
S-orthonitrothiOphenyl- -cysteine

N-tris(hydroxymethyl)methyl-2-
aminoethane sulfonic acid

xi

ORGANIZATION

This dissertation is divided into four sections. The
first section is composed of a brief description of aspects
of pyridoxal-P catalysis pertinent to this thesis and a
literature survey on tryptophanase from Eschenichia colt
B/1t7-A. The next three sections contain the results of
investigations into the mechanism of tryptophanase catalysis
and activation by monovalent cations. These three sections
are presented in a form similar to that required for publi-
cation in most scientific journals. An appendix containing
the derivations of equations used in the computer-assisted
fitting of experimental data is included at the end of the

text.

xii

Section I

INTRODUCTION

INTRODUCTION

A Brief Overview of Pyridoxal-Phosphate Catalysis:

The reaction of pyridoxal-P with amino acids is used
today as a model system by students interested in the mechan-
ism of enzyme catalysis. Pyridoxal-P achieved recognition
as an important biological catalyst when the first mechanistic
hypotheses regarding the chemistry of this compound were
advanced by Snell (l) and Braunstein (2) in 1958. Since that
time, pyridoxal-P has been shown to function as a coenzyme
for a large number of enzymes. In addition, pyridoxal and
pyridoxal-P, in the complete absence of enzymes, were Shown
to catalyze a,8-elimination reactions of amino acid substrates
as well as various other reactions such as transamination,
decarboxylation, and racemization carried out by enzymes
which require this cofactor (3).

The mode of action of pyridoxal-P can be most easily
visualized if one assumes that the coenzyme replaces the
a-amino group of amino acid substrates with a group which is
electronically the equivalent of an adjacent carbonyl (4).
This configuration allows for electron withdrawal from the
a-carbon toward the carbonyl group, in the case of model
structure I as shown in Scheme I, or, in the case of pyri-
doxal-P, toward the electronegative pyridinium nitrogen as

outlined in Scheme 1, structure II.

1

Pyridoxal-P generally participates in reactions by pro-
moting electron withdrawal from the uncarbon of the bound
amino acid leading to the cleavage of the bonds designated
a, b, and c in structure II. The role of the enzyme molecule,
then, is to enhance the rate and to confer Specificity to
ensure that a single set of products is made instead of the
several possible sets which can form in the absence of pro-
tein. As an example of pyridoxal-P catalysis, let us consi-
der an a,B-elimination reaction. In this case, an amino acid
substrate with a leaving group, X, on the B-carbon interacts
to form a Schiff's base with the coenzyme via a transaldimin-
ation reaction with the pyridoxal-P bound to an enzyme through
an s-amino group of lysine. (structure I, Scheme II). Loss
of the a-proton, presumably assisted by a basic group at the
enzyme active site, leads to a quinonoid intermediate 11
which characteristically absorbs at around 500 nm. This
intermediate can then lose X as shown in Scheme II to form
a bound a-amino-acrylate complex, III. The reaction is
completed following a second transaldimination reaction,
this time with the c-amino group of lysine acting as a
nucleOphile at the carbonyl carbon of the imine, to release
the imino acid (5) which subsequently undergoes non-enzymatic

hydrolysis.

 

 

 

 

 

0 __ a
fiz COO RV<€76 COO
of) \R I ijW?
0"
{L I
SCHEME 1 N
H“ 11
i '.
NH2 B».H
X-CHZ—{C- coo
{NtH ..___-'——*'__
o-
1 u
N I
+1+

 

 

 

 

 

 

SCHEME I I

A. Introduction - TryptOphanase was first recognized by
Happold and Hoyle in 1935 (6) as the enzyme responsible for
the production of indole in bacterial cultures. Subsequently,
Happold and Struyvenberg (7) demonstrated the requirement for
NH,+, K+ or Rb+ for enzymatic activity and that Na’ and Li+
were apparently inhibitory. Wood, Gunsalus and Umbreit (8)
discovered that tryptOphanase required the coenzyme pyri-
doxal-P and that pyruvate, indole and ammonia were formed
in stoichiometric amounts from tryptOphan. These and other
investigations led to the formulation of the basic reaction
catalyzed by tryptophanase:

tryptophanase

L - tryptOphan + H20 i indole + pyruvate + NH,
pyridgxal-P
K

 

A recent review by Snell (9) effectively summarizes most
of what was known about the enzyme up to 1975 and reviews by
Happold (10) and Nada (11) cover early aspects of these
studies.

B. Source of the enzyme - TryptOphanase is induced in a
wide variety of bacteria (9) where it apparently plays a role
in the catabolism of tryptOphan. However, the enzyme is
formed in variable amounts and differs in physical and
catalytic properties depending on the bacterial source (9).

In order to consistently obtain high yields of trypto-
phanase Newton and Snell (12) develOped a constitutive Strain

of Escheaichia colt called B/1t7-A. Since this mutant lacks

the genes for tryptOphan synthetase (l3) tryptophan for
protein synthesis must be obtained by reversal of reaction
1. This undoubtedly contributes to the high yield of
tryptOphanase obtained (up to 10% of the soluble protein)
when these cells are cultured in the presence of indole.

C. Structural prOperties - TryptOphanase from E. c021
B/1t7-A has a molecular weight of 220,000 and is composed
of four identical subunits (14). It is a pyridoxal-P
dependent enzyme with one coenzyme moiety bound per subunit
in an azomethine linkage to an e-amino group of a lysine
residue at the active site. As with other pyridoxal-P
enzymes, the coenzyme can be resolved from tryptOphanase
to give apoenzyme by the addition of reagents such as peni-
cillamine or cysteine which form thiazolidine derivatives
with pyridoxal-P.

Snell and coworkers (14) carried out extensive ultra-
centrifugation studies on both apo- and holoenzyme and
showed that the conversion of apo- to holoenzyme is accom-
panied by a large conformational change. This conformational
change resulted in a more compact structure which may account
for the greater stability of holoenzyme to denaturation by
sodium dodecyl sulfate (14), heat (15) and changes in pH.
These studies also revealed that the apoenzyme in the presence
of Na+ or K+ but not imidazole undergoes a concentration-
dependent dissociation into dimers as the temperature is
decreased below 20°C (14). In his review article (9), Snell

points out that since Na+ and K‘ behave in a similar fashion

with respect to dissociation of the apoenzyme, it is unlikely
that this effect is related to the large difference in the
ability of these cations to activate tryptOphanase catalyti-
cally. In contrast to apoenzyme, tetrameric holoenzyme
remained intact throughout the entire temperature range

3 - 25°C.

Toraya et a2. (16) compared by gel filtration studies
the effect of various monovalent cations on the binding of
the coenzyme and their effectiveness as activators. They
found that NH,+, KI and Rb+, which are good activators,
enhanced the binding constant for coenzyme. Enzyme in the
presence of the poor activators Cs‘, Na+ and Li+ demonstrated
a lower affinity for pyridoxal-P. The dissociation constant
was 31 uM and 1.8 uM in the presence of Na+ and K*, respec-
tively.

A possible criticism of this study is that the concen-
trations of cations used (0.1 M) may not have been saturating.
Suelter at at. (17) have shown that the activation constants
for the various cations vary from 54 mM in the case of Li+
to 0.23 mM for NH.I (Table I). The failure to observe
activity in these studies may be partially attributable to
low cation concentrations rather than to intrinsic differ-

ences in pyridoxal-P binding.

Table I

Activating Constants for Monovalent Cations

CATION KD (mM)
Lithium 54 t 11.6
Sodium 40 t 0.06
Potassium 1.44 2 0.06
Thallium (I) 0.95 1 0.1
Ammonium 0.23 1 0.01
Rubidium 3.5 t 0.3

Cesium 14.6 t 2.6

D. Spectral preperties - HolotryptOphanase exhibits
three absorption maxima centered at 278 nm, 337 nm and
420 nm. The peak at 278 nm is due to protein while the
peaks at 337 nm and 420 nm are contributed by the bound
coenzyme. On the basis of model studies (18) pyridoxal-P
aldimines absorbing in the wavelength range 410 nm - 430 nm
are usually associated with the resonance forms identified
as I in Scheme III. Since changes in the state of protona-
tion at the pyridinium nitrogen give rise to only small
changes in absorbance and positions of absorbance maxima (19)
the state of protonation at the pyridinium nitrogen for
most enzymes is unclear. Form Il may exist in some cases.
The coenzyme structure giving rise to the 337 nm band in
tryptOphanase is probably 111 as suggested by Davis and
Metzler (20) and Snell (9).

Marina and Snell (21) demonstrated that the relative
intensities of the interconvertible 337 nm and 420 nm bands
of tryptOphanase depended on both pH and the nature of the
monovalent cation in the medium. At low pH values the
420 nm form of the enzyme predominated. As the pH was
increased, absorbance at 337 nm increased at the expense
of 420 nm absorbance. According to Marina and Snell (21)
the change in 337 nm and 420 nm absorbances as a function
of pH describe a single titration curve with an apparent

pKa of 7.2 indicative of a single proton process. These

 

SCHEME III

10

authors also showed that in the absence of monovalent cations,
that is, in imidazole-HCI buffer, the bound coenzyme was
entirely in a form which absorbed at 420 nm. The addition
of 0.1 M Na+ caused an increase at 420 nm which may be
attributable to increased binding of pyridoxal-P. Neither
of these spectra changed significantly between pH 7.0 and
9.0. When 0.1 M K+ replaced Na+ as the cation at pH 8.0,

a dramatic change was observed in the spectrum in which the
337 nm form of the enzyme became the dominant Species. They
associated the 337 nm form of tryptOphanase with activity
because this was the form that predominated at pH values
above 8.0 where the enzyme exhibited its greatest apparent
activity. The observation that effective monovalent cation
activators promoted the formation of the 337 nm form, while
a poor activator, NaI, did not, also appeared to be consis-
tent with this interpretation.

Although other coenzyme structures could give rise to
absorbance at 337 nm, Davis and Metzler (20) argue that
Scheme III serves to explain the effects of pH and monovalent
cations on the absorption spectrum of tryptOphanase observed
by Marina and Snell (21), if it is assumed that dissociation
of a proton from I, with an apparent pKa of 7.2, ultimately
gives structure III. The structures II and III would be
favored at high pH values and in a hydrophobic environment.

The increased amount of 337 nm absorbance in the presence

11

of effective monovalent cation activators is consistent with
the data of Toraya at at. (16) showing that the coenzyme is
bound more tightly, and presumably in a less polar environ-
ment, in the presence of these cations.

E. Mechanism of tryptophanase catalysis - A mechanism
of tryptophanase catalysis consistent with the available
data was outlined by Snell (9) in his review article. This
mechanism is highly schematic in the sense that several
intermediates (e.g. those which occur during the transaldim-
ination reactions) are not shown. Likewise, Snell mentions
that the exact ionic forms of the coenzyme involved at each
step are not known.

The first step in this preposed reaction sequence involves
the conversion of inactive enzyme, represented by structure
I in Scheme IV, absorbing at 420 nm, to an active form of
the enzyme (II) which absorbs at 337 nm. As mentioned,
Marina and Snell (21) obtained an apparent pKa value of
7.2 for this process in the presence of potassium.

The second step involves the formation of the Michaelis
complex between the enzyme and a suitable amino acid sub-
strate or inhibitor via a transaldimination reaction. The
third step is the loss of the a-proton from the bound amino
acid to form the quinonoid intermediate IV which character-
istically exhibits intense absorption at approximately

500 nm (21). The reaction with dead-end inhibitors staps

 

 

12

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RCHZCHCOO'
NH2
?

NHZ H NH?
RCHzé}coo :H+ R- CHaC- coo
N r 4 N
if I)

2' )
... l "N I
3+ ”1 I. N
A
:RH
V
II+CH c'cooQNH: * ‘ NH2 CH2=C-COO
l
/N
/
|
3+ v

SCHEME IV

13

at this point. In the case of substrates, that is amino
acids with labilizable B-substituents, this absorption band
disappears as substrate is depleted. Experiments in szo
and ’H20 (21) in the presence of the inhibitor L-alanine or
substrates confirmed that the a-proton of alanine is labil-
ized during quinonoid formation and that o-proton loss occurs
at a rate faster than elimination of the B-substituent when
substrates other than S-orthonitrophenyl-L-cysteine (SOPC)
(21) are used. Suelter and Snell (17) demonstrated that no
tritium was incorporated into unreacted SOPC and concluded
from this that loss of the a-proton was rate-limiting for
the reaction with this artificial substrate.

The fourth and fifth Steps in Scheme IV represent the
loss of the B-substituent of the substrate, the ultimate
regeneration of the active enzyme and product formation.
Based on their results, Hillebrand at at. (5) suggested that
a-aminoacrylate was released from the active site either
as a-iminOprOpionate or the carbinolamine of pyruvate when
SOPC was the substrate.

It is clear from this summary of the mechanism of
tryptOphanase catalysis that much work remains to be done
in elucidating the forms of pyridoxal-P which participate
in the catalytic process and the effects of monovalent
cations on the Spectral and kinetic prOperties of the

enzyme.

10.

11.

12.

13.

14.

15.

16.

14

REFERENCES

Snell, E. E. (1958) Vitamins and Hormones, 16, 77-125.

 

Braunstein, A. E. (1960) in The Enzymes (Boyer, P. D.,
Lardy, H., and Myrback, K., eds.)‘Vol. 2, pp. 113-184,
Academic Press, New York.

 

Metzler, D. E. and Snell, E. E. (1952) J. Biol. Chem.
198, 363-373.

 

Metzler, D. E., (1977) Biochemistry; The Chemical
Reactions of Living Cells, Academic'Press,*Neinork.

 

 

Hillebrand, G. C., Dye, J. L., and Suelter, C. H. (1979)
Biochem. 18, 1751-1755.

Happold, F. C., and Hoyle, L. (1935) Biochem. J. 22,
1918-1926.

 

Happold, F. C., and Struyvenberg, A. (1954) Biochem. J.
58, 379-382.

 

Wood, W. A., Gunsalus, I. C., and Umbreit, W. W. (1947)
J. Biol. Chem. 170, 313-321.

 

Snell, E. E. (1975) Adv. Enzymol. Relat. Areas Mol.
Biol. 42, 287-333.

 

Happold, F. C. (1950) Adv. Enzymol. Relat. Subj. Biochem.
12, 51-82.

Wada, H. (1964) Tryptophan Metabolism Vol. 1, Sekai Hoken
Tsushinsha, Ltd.,O§aka, Japan, 77-92.

 

Newton, W. A., and Snell, E. E. (1962) Proc. Nat. Acad.
SCio UoSvo' 4—8’ 1431'14390

 

Yanofsky, C., and Crawford, I. P. (1959) Proc. Nat. Acad.
Sci. U.S.A., 15, 1016-1026.

 

Morino, Y., and Snell, E. E. (1967) J. Biol. Chem., 242
5591-5601.

 

Raibaud, 0., and Goldberg, M. E. (1973) J. Biol. Chem.
248, 3451-3455.

Toraya, T., Nihira, T., and Fukui, S. (1976) Eur. J.
Biochem. 62, 411-419.

15

References - continued

17.

18.

19.

20.

21.

22.

Suelter, C. H., and Snell, E. E. (1977) J. Biol. Chem.
252, 1852-1857.

 

Heinert, D., and Martell, A. E. (1962) J. Amer. Chem.
SOC. 8—4-’ 3257-3263.

 

Johnson, R. J., and Metzler, D. E. (1970) Methods
Enzymol. 18A, 433-471.

Davis, L., and Metzler, D. E. (1972) in The Enzymes
(Boyer, P. D. ed.) Vol. 7, pp. 33-74.

 

Morino, Y., and Snell E. E., (1967) J. Biol. Chem. 242,
2800-2809.

Suelter, C. H., Wang, J., and Snell, E. E. (1976) FEBS
Lett. 66, 230-232.

SECTION II

EFFECTS OF pH AND MDNOVALENT CATIONS ON THE
SPECTRAL AND KINETIC PROPERTIES OF TRYPTOPHANASE

16

TryptOphanase (E.C. 4.1.99.1) from Eacheaichia colt
B/1t7-A is a member of a group of pyridoxal-P dependent
enzymes catalyzing o,B-elimination reactions of amino acid
substrates (1). As with other members of this group, trypto-
phanase requires the presence of monovalent cations for
Optimum catalytic activity (2,3,4). The enzyme is sensitive
to the nature and concentration of the monovalent cation and
the pH of the environment (4,5). This sensitivity is mani-
fested in marked changes in the absorption spectrum and
alterations in kinetic behavior. This paper presents stu-
dies on the effects of pH and monovalent cations on trypto-
phanase in the presence and absence of substrates and
inhibitors which were undertaken in an attempt to gain
insight into the role of monovalent cations in this reaction
and the functions of ionizable groups on the enzyme and/or
pyridoxal-P involved in substrate binding and turnover.

A simple model is presented which suggests that mono-
valent cations may activate tryptophanase by altering the
equilibrium between a catalytically inactive conformation,

and a catalytically active conformation.

EXPERIMENTAL PROCEDURES

 

Materials.- RbCl, CsCl and LiCl were obtained from

 

Ventron, Danvers, Mass. as ultrapure products. NH.C1 and
KCl were Mallinckrodt, analytical reagents. Pyridoxal-5'-

phOSphate, N-2-hydroxyethy1piperazine prOpane sulfonic

17

acid (Epps), N-tris(Hydroxymethy1)methyl-Z-aminoethane
sulfonic acid (Tes), N,N-bis(2-hydroxyethyl)glycine (Bicine)
and 2(N-Morpholino)ethane sulfonic acid (Mes) were obtained
from Sigma Chemical Co. 1,3-bis[tris-(hydroxymethyl)methyl-
amino]pr0pane (Bis-tris-propane) was from Cal Biochem.
(CH3)~NCL from Aldrich Chemical Company was recrystallized
from N-prOpyl alcohol before use. (CH,)~NOH was prepared
fresh before use by passage of recrystallized (CH,).NCL over
Dowex-l-OH.

TryptOphanase - TryptOphanase from Eachenichia c022
B/lt7-A was prepared as described by Watanabe and Snell (6)
including the modifications of Suelter at at. (7). Protein
was judged to be greater than 95% tryptOphanase by polyacryl-
amide gel electrOphoresis. Holoenzyme was prepared from
stock apoenzyme by incubation in 0.1 M potassium phosphate,
pH 8.0, 7% (NH.)SO., 1 mM EDTA, 0.2 mM pyridoxal-P, 20 mM
dithiothreitol (DTT) for 1 hour at 37°C. Occasionally, acti-
vation was achieved in the same buffer at 50°C for 15 min as
suggested by Hdgberg-Raibaud at at. (8). The enzyme had a
Specific activity of 40 - 50 umol°min"-mg'l when assayed
with 0.6 mM S-orthonitrOphenyl-L-cysteine (SOPC) in 50 mM
potassium phosphate, pH 8.0, 50 mM KCl, at 30°C (9).
Activating monovalent cations were removed from enzyme
solutions by extensive dialysis. Protein concentration was
determined spectrophotometrically using c,,,-0.795 ml mg"1

cm'l(4).

18

Kinetics - Kinetic constants obtained in the presence
of 0.5 M KCl and 0.85 M LiCl at various pH values were deter-
mined from initial velocities at five SOPC concentrations
ranging from 0.056 mM to 0.604 mM in 25 mM (CH,),N-Tes or
(CH3).N-Bicine, 0.1 mM pyridoxal-P and 2 mM [(CH,),N],-EDTA.
Values for Km and Vmax as a function of pH in 0.2 M KCl were
obtained by analysis of complete reaction progress curves.
Absorbance versus time data were fit to the Michaelis-Menten
equation with the nonlinear curve fitting program KINFIT (10).
The following initial concentrations of SOPC were used:
1.35 mM for pH values 6.60, 6.76; 7.00 and 7.23; 1.10 mM for
pH 7.40 and 0.42 mM for all other pH values. The remainder
of the assay mix consisted of .025 M (CH,),N-Mes or
(CH,)~N-Epps, 0.2 M KCl, 20 uM pyridoxal-P and 2 mM EDTA.

Anion Inhibition - TryptOphanase was dialyzed against

 

5 mM K-Epps, pH 8.0, 40 mM KCl, 1 mM EDTA, 0.1 mM pyridoxal-P
and 0.2 mM DTT. Five ul of this enzyme solution (1.5 mg ml")
was added to a 1 ml assay consisting of 0.6 mM SOPC, 25 mM
K-Epps, pH 8.0, 1 mM EDTA and the indicated potassium salt

at a concentration of 0.2 M. All assays were carried out

at 30°C. Km and Vmax were obtained by analysis of complete
reaction progress curves.

Spectral Titrations - Dissociation constants for the

 

conversion of the 420 nm form to the 337 nm form of trypto-
phanase in the presence of various monovalent cations were

obtained after extensive dialysis at 4°C against 5 mM

19

(CH,),N-Epps, pH 8.0 containing the indicated concentration
of monovalent cation, 0.1 mM pyridoxal-P, 1.0 mM [(CH,).N]z-
EDTA, and 0.2 mM DTT was added to nine tenths m1 of .025 M
(CH3).N-Epps, pHi, 0.1 mM pyridoxal-P and 1.0 mM [(CH;).N]2-
EDTA. The final pH value of each protein solution was
measured at 30°C on a Sargent-Welch digital pH meter subse-
quent to measuring the absorbances at 337 nm and 420 nm.
The composition of the reference was identical to the sample
with the exception that one-tenth ml of a dialysate was
substituted for the tryptOphanase solution. Absorbance
measurements were made at 30°C with a Beckman DU spectro-
photometer equipped with a thermostated cell compartment.

pH Stability of the Enzyme in the Presence of Various

-1 .
Monovalent Cations - TryptOphanase at 2 mg ml was 1ncubated

 

at 30°C for a period of 10 minutes in 0.025 M (CH,),N-Epps,
in the pH range 6 - 10, containing either 0.1 M KCl, 0.5 M
RbCl, 0.5 M CsCl or 0.6 M LiCl, 0.1 mM pyridoxal-P, 1 mM
[(CH;).N]2-EDTA and 0.02 mM DTT. Aliquots were then removed
and assayed at 30° C in the Standard SOPC reaction mixture.
Titration of tryptOphanase with L-alanine as a function
gf_p§ - One tenth ml of tryptOphanase (approximately 25 mg
ml") in 1 mM (CH3).N-Epps, pH 8.0, 0.2 M KCl, 1 mM EDTA,
0.1 mM pyridoxal-P, 0.2 mM DTT was added to nine tenths m1
of 0.025 M (CH3),N-Epps, pHi, 0.2 M KCl, 1 mM EDTA, 0.1 mM
pyridoxal-P in a quartz cuvet. The composition of the refer-

ence was identical to the sample with the exception that one

20

tenth ml of the dialysate was substituted for the trypto-
phanase solution. Aliquots of 1.1 M L-alanine, 0.1 mM
pyridoxal-P, 0.2 M KCl, 1 mM EDTA at pHi, were added to
each cuvet and the absorbance was measured at 502 nm. The
final pH of each titration mixture was measured subsequent
to addition of the last aliquot of alanine.

Treatment of Data - All kinetic and titration data
were fitted to the apprOpriate mathematical function with

the non-linear curve fitting program KINFIT (10).

21
RESULTS

Buffer Effects and Stability - To rule out Spurious
buffer effects in these pH studies, the initial velocities
of tryptophanase in several overlapping buffer systems were
investigated (Figure l). The initial velocities in the over-
lapping pH regions of Mes, Tes, Bicine and Epps were similar
and gave an apparent pH Optimum of 8.3 under these conditions.
Bis-tris-prOpane tended to inhibit at low pH values.

From results not shown it was found that tryptophanase
under conditions described in Materials and Methods lost
less than 5% of its activity when incubated with KCl or RbCl
over the pH range 6.2 - 9.5. Enzyme in 0.5 M CsCl or
0.6 M LiCl retained the same stability over a smaller range
of pH 6.5 - 9.0.

Spectral Titrations of Tryptophanase in the Presence of
Various Monovalent Cations - Previous studies by Morino and
Snell (4) showed a pH dependent interconversion of the 420 nm
and 337 nm bands of tryptophanase. These studies were
extended to determine whether the monovalent cations, NH.+,
KI, Rb+, Cs+ and Li+ affected the pKa' for this intercon-
version (Figure 2 and Table 1). Complete titration curves
for Cs+ and Li+ could not be obtained since the enzyme was
not stable for sufficient time at the extreme pH values.
Consequently, the pKa' values given for Cs+ and Li* in

Table I are approximations.

22

F1 ure 1. Effect of various buffers on tryptOphanase
activity. I 1 ml assay consisted of 0.6 mM S-orthonitro-

phenyl-L-cysteine, 0.025 mM buffer at pHi, 0.2 m KCl, 10 uM
pyridoxal-P, 1 mM EDTA and 5 ug of purified tryptOphanase.
Assays were carried out at 30° C in Mes (O), Tes (A),

Epps (+), Bicine (0), and Bis-tris prOpane (X).

23

 

 

9.60

 

 

(\9 z
I\. nu
.. + S
X H
A DI
nu
E
I : I7
0
o : 0
cu
0 u
1 J D-W
8.8 8.9. 8. ow 8.26

>HmouomM> JchHzH

FIGURE 1

24

Fi ure 2. Plots of the absorbance of tryptOphanase at
420 nm 56) and 337 nm (A) measured as a function of pH as
described in Materials and Methods. A) 0.025 M NH.C1;
B) 0.1 M KCl; C) 0.2 M KCl; D) 0.5 M KCl; E) 0.5 M RbCl;
F) 0.5 M CSCl; G) 0.6 M LiCl. The solid lines in each
graph were calculated using equation 1 with the parameters
listed in Table I.

25

 

 

9.50

 

 

A. 0 A

.4 0

cm

mu

1 Cu

.. . .7.

.1 0

cm

8.2 8.8; 8.11. 8....5
OOHXHOZ\MQZ¢mmowm¢H

PH

2A

FIGURE

26

 

C'I

 

11.00

9.00

PH

7.00

 

oo.NH i0

8. Ho 8
OOfiXHOZ\muZ¢mmowm¢H

 

"35.00

23

FIGURE

 

0

 

 

FI GURE

r
8.60

1
9.60

100

JX

10.00

(HBSURBRNCE/MG

4.00

28

 

13.00

 

'1'

 

 

6.

7'.75 81.75 91.75
PH

FIGURE 2D

00

13-
I
P

l

10.00

CE/MGJX100

BSORBRN
7(00

 

29

I!)

'3

t.’

 

 

7140 8.40 9140
PH

FI GURE 2E

30

 

12.00

 

100

 

 

 

 

”6.50 8:50 10.50
PH

FIGURE 2F

12.50

13.00

100

IX

10.00

(HBSORBRNCE/MG
7.00

 

4.00

31

 

6.50 8.50

99 ,,u 4

10.50
PH

FIGURE 26

 

112.50

32

.:«auno on onnwmmon no: one: muooqummoou :oMuuofluxo omega
ouaanoaau on «deflowmmsm «new .m mm o>onm oexueo on» we xumawnaumcw on» mo omamoom .n

.mponooz pen mnuwuouaz
:« poewauao mu newuau sumo umefiuma mwmxnmwv o>wm=ouxo uouma :omuauwmouo oaxuco

eoao mo .8 mg ouaeamoea snowmauon :5 cm .Hou :3 on .uaom 2a 8.8 a“ sau>uuua ummauonm .a

 

 

 

n onN n coco ms.m me o.o +wq
A -s~ a mean mm.m an m.o +mo
mm H acom mw H fimvN on H mowN ~m u Hs~u No.o w om.w on m.o
mm H vam mm H mth an « “NAN mm a exec no.0 a no.» em m.c +nm
as H caoo me A ammN mu H eou~ Nm H wean no.c H co.m vm m.o +M
mm H ommm on H hmvN em H oumm em « ammo mc.o H on.» on N.o +x
mNH w wamm aHH u emn~ enH H cch “an n mono 09.: a vu.m cm H.c +3
ona H ws~o ~u~ w vuam n~H H mmnm mad H cn~o ce.c « mm.u ve mwc.o +.=z
< =< =< Hzg
...u ...u ...u ...u .aeq a<m company one<u
Icoucou
.ommcmgnouoxuu mo mauom E: snn pen a: oNv 0:9 mo
cowmuo>eouuouew ecu you «an acouuoom may :0 meowumu woodm>ocos mo uuomwo 039 “H agony

33

The titration curves for each cation, NH.+, K+ and RbI,
described by the decrease in absorbance at 420 nm and the
increase in absorbance at 337 nm were fitted by the nonlinear
curve fitting program KINFIT, as a multiple-data-set* to

equation 1, previously given by Johnson and Metzler (ll),

eobs ' €A[1°(pH-pK)] * EHA

 

[1 + 10(PH'PK)] (1)

where EA - calculated molar absorption coefficients at 337 nm
or 420 nm at infinite pH, EHA - calculated molar absorption
coefficients at 337 nm or 420 nm at zero pH, and Eobs .
observed molar absorption coefficients at 337 nm or 420 nm.
Thus, five parameters, SAH°”, cA’°’, eAH“z°, eA°2° and the
common parameter for both sets of curves, pKa', were adjusted
to obtain the best fit of the data for each cation.

The data in Table I Show that pKa' depends both on the
cation and on the ionic strength. If one were to assume that
pKa' for NH.+ would be higher if the ionic strength were
increased, as was observed with K+, then the pKa' values
obtained in NH.*, K’, and Rb+ are essentially identical but
different from those observed with Cs+ and Li+. Although the

data presented here have been analyzed in terms of a single

ionizing group, it should be pointed out that recent kinetic

 

* When two or more sets of data, each being fitted to a
separate function, share at least one adjustable parameter,
the data may be fitted simultaneously with KINFIT as a
so-called multiple-data-set.

34

data has shown this interconversion to be a more complex
process (12). On the other hand, it is clear that the pKa'
value for the 337 nm - 420 nm interconversion depends on the
monovalent cation, that is, the more effective the cation

is as an activator, the lower the observed pKa'. The cal-
culated extinction coefficients at 337 nm and 420 nm of the
enzyme at high and low pH values were essentially the same

I and RbI. The data with Cs+ and Li‘ are not as

in NH.*, K
complete but the extinction coefficients at 337 nm and 420 nm
at low pH values are comparable to those observed with NH.+,
KI, and RbI. The observed variation in the extinction
coefficients can be accounted for in terms of the Specific
activity of each enzyme preparation after the extensive
dialysis used to exchange the cations. Generally, the higher
the Specific activity the higher the €AH~20 and SA“’° and

the lower the SAH’37 and SA”’. On the other hand, the

values observed for [(cAH“2°-

SA“2°)/Specific activity] and
[(cAH’37-SA°”)/Specific activity] are constant, being
82 1 S and 63 t 6, respectively.

As indicated in Figure 3, Vmax, using SOPC as a sub-
strate, decreases as the pKa' of the 420 nm - 337 nm
spectral transition is increased. It is intriguing to

note that 0.5 M KCl causes an increase of 0.35 units in

pKa as compared to 0.1 M KCl and a 20% decrease in Vmax.

35

Figure 3. Relationship between Vmax for the degrad-
ation of SOPC and pKa' for the interconversion of the
420 nm and 337 nm forms of tryptOphanase.

 

36

I l

 

80.00

I

40 .00

l

(MICROMUL/MIN'MG]
20.00

VMRX
00

0 0.02514 NH4CI

  

0.1M KCI A 0.2M KCI

     
   
  

0.5M RbCI + 0.5M KCI

0 0.5M CSC'I

0.6M LiC'I X

 

 

 

\P

I

.50 8150 9.50
PK

FIGURE 3

10.50

37

Anion Inhibition of TPase - To determine if the inhibi-
tion by high concentrations of KCl was due to the presence
of an anion binding site on the enzyme as is the case with
serine transhydroxymethylase (13), Km and Vmax were deter-
mined in 0.2 M KCl, K-acetate, KBr, KF and KNO,. Only KNO3
appeared to inhibit, acting in a competitive manner with
substrate. Km was increased from 0.13 mM to 0.47 mM. Fluor-
ide, which might be expected to bind strongly to an anion
binding site tended to decrease both Km and Vmax, but by
less than 10%.

pH Dependence of the Interaction of L-Alanine with

Trypt0phanase - L-alanine, a competitive inhibitor of trypto-

 

phanase, interacts with bound pyridoxal-P to form a quinonoid
absorbing maximally at 502 nm (4). The dissociation constant,

K for this interaction was examined over the range pH 6.5 -

D,
9.0 (Figure 4). The data were analyzed in terms of Scheme I
which describes the binding of a competitive inhibitor which
has no dissociable group in the pH range of interest with

enzyme which has a dissociable group in this pH range.

 

 

Ki

E4 rEI

I
+ +
K 1 1
Scheme 1 E H H EH

I

EH~ *‘EHI

 

38

.HH «Hoop cm ocflcmaa you :o>ww muouosmumm on» momma
axou no N sewumsvo no“: boondouHoo no: ocma och .ocucm «-4 how
.92 .u:mum=ou :owuwwuommmp ogu mo oucovcoooc :9 ans .v ow: «m

 
 

39

v ouzmwu

mmd 0N0 mm.» mmw
_ _ _ ood

 

00..
ova

 

 

 

_ _ ooN

 

40

The apparent dissociation constant, KD, at any pH is
given by equation 2, taken from Cleland (14), where EH and

E represent protonated and unprotonated enzyme, respectively.

Ki (1 + [H*]/K )
KD . + Pili- (2)
(1 + [H l/KEH)

 

Analysis of the data in terms of this model using KINFIT (10)
gave the following results: PKE - 8.47 t 0.10, Ki 8 9.1 t
0.8 mM. pKEH . 6.7 and Kj - 0.6 M. KEH and Kj are not well
defined due to the experimental difficulty of saturating the
enzyme with alanine at low pH values where the concentration
of alanine would need to approach the value of 0.6 M to
achieve half saturation of the enzyme. These results do
imply, however, that under normal experimental conditions
essentially no inhibitor binds to the EH form of the enzyme.

Kinetics of SOPC Degradation - Kinetic studies in the
presence of 0.2 M KCl, 0.5 M KCl and 0.85 M LiCl reveal that
Vmax is constant with pH and that the Km for SOPC varies
with pH in a similar fashion under all three conditions. A
plot of log (V/K) versus pH (Figure 5) reveals two downward
inflection points. Since V/K is the apparent first order
rate constant for the interaction of enzyme with substrate
(15), these inflections imply that ionizations which occur
on the free enzyme and/or free substrate affect binding.
Data for the binding of alanine to tryptOphanase as a

function of pH showed that for all practical purposes only

41

Fi ure 5. Variation of the affinity of tryptophanase
for SOPC a d the maximal velocity (V) with pH. The kinetic
parameters were determined as described in Materials and
Methods in the presence of A) 0. 2 M KCl; B) 0. 5 M KCl;

C) 0.85 M LiCl. The amount of purified tryptophanase used
in each case was: A) 10 ul of 1.3 mg ml , B) 5 ul of

1.0 mg ml 1 C) 10 ul of 8. 8 mg ml The units of (V/K)
are min" and the units of V are umol min' mg ‘. The

lines were calculated with the parameters listed in Table
II.

42

 

 

 

 

om.N

 

ov.H

 

 

 

9'50

8.50

PH

Figure 5A

I

7.50

 

 

oo.N

"6.50

43

 

 

 

oo.m

om.N
Hx\>g

CM:
Cy:

~4&

 

 

 

 

9.50

 

 

A
nU
Cu
I A Tom
m. ”H”
DI
o nu
1 PM
1 Ifil
.w
6
my nU
CU
comm so #5
H>HOOJ

Figure SB

44

 

 

 

 

oo.m om.~

 

 

 

 

9.50

8.50

PH

Figure 5C

7.50

 

8.N
:5

CDC)
_JC)

 

”5.50

45

the inhibitor in the zwitterionic form (pKa of a-amino
group - 9.6) binds to unprotonated enzyme, E. Since the
pKa of the a-amino group of SOPC was determined to be
8.44 t 0.03 in 0.2 M KCl and 8.40 t 0.04 in 0.5 M KCl at
30° C it was assumed that ionization of this group also
dramatically lowered its affinity for the enzyme. If we
assume that dipolar SOPC binds to unprotonated enzyme we

arrive at the following mechanism:

S
, '0‘
ksopcI’E k3
E + SHéEs—‘E + P
KE 1H+ 2 Scheme II

EH
where S and SH refer to unprotonated and protonated forms

of SOPC, respectively. From steady-state considerations,
assuming Michaelis-Menten behavior and that the protonation-
deprotonation steps are in rapid equilibrium, one obtains

the following:

v - V

[Km/15mm + [WI/Km) + 1 ‘3)

 

where Km -(k2 + k,)/k,, and KB I (E)(H)/(EH).

46

After substituting an expression for [SH] in terms of 50'

total SOPC, we obtain

vso
Km (1 + IH*I/KE)(1 + Ksopc/IH*I) + 80

v.

(4)

 

where KSOPC is the dissociation constant for the a-amino
group of SOPC. Since V is independent of pH, no Michaelis
pH function (16) such as the ones appearing with Km in
equation 4 need be associated with this term.

All initial velocities obtained at various SOPC and
hydrogen ion concentrations were then fitted by using
equation 4 in program KINFIT To obtain pKB, Km and Vmax
in the presence of 0.2 M KCl, 0.5 M KCl and 0.85 M LiCl.

The kinetic data with SOPC and the alanine titration data

are summarized in Table 11.

DISCUSSION

 

The results presented in this paper strongly support
our previous conclusion that monovalent cations interact at
or near the catalytic center of tryptOphanase in such a way
that they either participate directly in the reaction or are
required for the critical alignment of one or more functional
groups necessary for catalysis (5). The spectral titrations

of tryptOphanase as a function of pH demonstrate that the

47

 

 

 

 

 

 

 

 

 

 

.Ime..-=ME .HoE: ohm xwe> mo mafia: one .9
.uaom taaoawe you so .5
.oco w.o w H.a 5.0 I- - o~.o a ue.w H02 2 ~.c ocwcmu<
- I- - ~.c H e noo.o a oHc.o mM.: a we.» Howq mm.c umom
-- - - ~.H N no Noo.c « e~o.c oH.c H we.» Ho: 2 m.o umom
- - I- m.H A an voc.o a vmo.o cH.o w mH.m Hum z ~.c omom
:m m :owumu noufinwscm no
Azev nu AZEV fix an nxme> NAZEU Ex an acon>oeozw oumuummomI

 

.=a mo :oMuucsm a mu ocwemHa-a saw: owococwac
may we coauNEHom on» one umom mo :owumpmumop on» new whouoEmumo owuonwx ”Hm manna

 

48

cation has a dramatic effect on the equilibrium between the
337 nm and 420 nm forms of the enzyme. It is difficult to
imagine that such large effects would be seen if the cation
were bound at a Site remote from the coenzyme.

These results are consistent with the scheme outlined
by Davis and Metzler (l) in which deprotonation of the
pyridinium nitrogen leads to a species absorbing at 337 nm.
This mechanism is reproduced in Scheme III with the addition
of a form corresponding to a conformer which exists in the
absence of cations. That such a conformer exists is evident
because in the absence of cations the enzyme is inactive
and absorbs entirely at 420 nm. It is assumed that the
coenzyme which gives rise to the inactive 420 nm absorption
is similar in structure to HIE...o or E~20 but is bound in
such a manner as to preclude interaction with groups at
the active site necessary for catalysis.

The different pKa' values for the spectral interconver-
sion and Vmax values observed in the presence of the various
cations are consistent with the assumption that the role of
the cation is to affect the equilibrium between two confor-
mers, Co, a catalytically incompetent form that would exist
in the absence of cations, and CM, a conformer promoted to
different extents by the various cations. By lowering the
value of K1 (Scheme III), the cation could increase Vmax and

simultaneously decrease the apparent pKa for conversion of

_ _ H mzuzuw

 

so
_
...8 I 354 s... 8- I 565‘ E: 81.... .65
an 2.. 52 z
m. z u z m +: +2
IIV IIV
m o .... ..o
z... m \ L
I 5 A.
0:73 37>. 05...:

 

so
_ ‘
Scout-4.354
.5.
“+2 ow AI” +2... am

50

420 nm absorption to an absorption with a maximum at 337 nm.
Essentially identical values for the extinction coefficients
of the various forms of the enzyme in the presence of NH.‘,
K+ and Rb+ support this scheme. It should be pointed out
that various conformations may exist under the general desig-
nations of Cu and CM. In fact, this is almost surely the case
for CM which demonstrates at least two absorption maxima indi-
cating different coenzyme environments. The fact that the
absorbance at 337 nm does not go to zero at zero pH may be
attributable to absorption contributed by the various 420 nm
absorbers. According to Scheme III, absorption at 420 nm
would not be expected to go to zero at high pH due to the pH-
independent equilibrium between structures E~zo and E337.

We have also shown that although Vmax is different in
K+ and Li+, it is independent of pH throughout the range
investigated for both cations. This indicates that the
cation does not act by altering the pKa' of the a-hydrogen
of the bound substrate molecule. If this were the case, one
might expect to see an inflection point due to this ioniza-
tion in the log V vs.,pH plot for SOPC degradation in Li+.

Of course, it is possible that this pKa' is sensitive to
the cation, but in all cases is below approximately 6.6.

The fact that the Li+ enzyme shows activity even at low
pH values despite its pKa' value of approximately 9.75 also
suggests that activity may be associated with a 420 nm form
of the coenzyme rather than the 337 nm form as argued by

Marina and Snell (4).

51

The affinity of enzyme for substrate as a function of
pH is reflected in the log (V/K) plots for SOPC or in the
case of the inhibitor, alanine, in the pKD v4. pH plot.

We have assumed for the analysis of the SOPC binding that

it is the protonated form of the substrate that binds to

an unprotonated enzyme. This is not evident from the SOPC
data alone because the pKa' for the a-amino group is

similar to the pKa' for the free enzyme. However, the pKa
for the L-alanine amino group is 9.6, and thus it is fully
protonated throughout most of the pH range where binding
studies were done. If we assume that alanine and SOPC

are similar in this respect, then substrate must be pro-
tonated for binding to occur. The pKa' obtained for free
enzyme involved in binding obtained by equilibrium titration
data with alanine and from kinetics data with SOPC (TableII)
are not believed to be significantly different.

Therefore, the equilibrium binding data and kinetics
data are consistent with a common model involving binding
of protonated a-amino acids to an unprotonated enzyme.
Binding of protonated alanine to unprotonated enzyme was
some 65 times more effective (Kj/Ki -6S.9 ) than binding to
protonated enzyme. Similar data could not be obtained
with SOPC because inhibition was observed at elevated levels

of SOPC (9). The value of pKEH - 6.7 corresponds to the

52

value 6.65 given by Marina and Snell (4) for the loss of the
alpha proton of L-alanine in the protein complex. Since
attempts to fit the alanine binding data to the mechanism
given by Morino and Snell (4) were not successful, we are
not able to confirm this assignment.

Since pKE is independent of the cation, the simplest
explanation is that this group is on the protein itself and
must be ionized for substrate to bind. However, the possi-
bility remains that the process is more complex and involves

more than one functional group.

10.

11.

12.

13.

14.

15.

16.

53

REFERENCES

Davis, L., and Metzler, D. E. (1972) in The Enzymes
(Boyer, Po Do, ed.) V01. 7’ pp. 33-740

 

Happold, F. C., and Struyvenberg, A. (1954) Biochem. J.
53, 379-382.

 

Newton, W. A., and Snell, E. E. (1964) Proc. Natl. Acad.
SC]... UoSvo 2, 382-389.

Morino, Y., and Snell, E. E. (1967) J. Biol. Chem. 242,
2800-2809.

 

Suelter, C. H., and Snell, E. E. (1977) J. Biol. Chem.
252, 1852-1857.

 

Watanabe, T., and Snell, E. E. (1972) Proc. Natl. Acad.
SCio UoSvo ‘6_9-, 1086'10900

Suelter, C. H., Wang, J., and Snell, E. E. (1977) Anal.
Biochem. 16, 221-232.

Hdgberg-Raibaud. A., Raibaud, O., and Goldberg, M. E.
(1975) J. Biol. Chem. 250, 3352-3358.

Suelter, C. H., Wang, J., and Snell, E. E. (1976) FEBS
LBtt. £9. 230-2320

Dye, J. L., and Nicely, V. A. (1971) J. Chem. Ed. 18.
44 3-448 0

Johnson, R. J., and Metzler, D. E. (1970) Methods Enzymol.
18A, 433-471.

June, D. S., Kennedy, 8., Pierce, T. H., Elias, S. V.,
Halaka, F., Behbahani-Nejad, 1., El-Bayoumi, A., Suelter,
C. H., and Dye, J. L. (1979) J. Amer. Chem. Soc. 121.
2218-2219.

Schirch, L., and Diller, A. (1971) J. Biol. Chem. 246,
3961-3966.

Cleland, W. W. (1977) Adv. Enzymol. Relat. Areas Mol.
Biol. 15, 273-387.

Cleland, W. W. (1970) in The Enzymes (Boyer, P. D. ed.)
Vol. 2, pp. 1-65, Academic Press, New York.

Dixon, M., and Webb, E. C. (1964) Enzymes, 2nd edition,
Academic Press, New York.

SECTION III

STUDIES ON THE KINETICS AND MECHANISM OF THE pH-DEPENDENT
INTERCONVERSION OF THE SPECTRAL FORMS OF TRYPTOPHANASE

54

TryptOphanase from Escheaichia c022 B/lt7-A is a
pyridoxal-P dependent enzyme which catalyzes a,B-elimination
reactions Of amino acid substrates and requires monovalent
cations for Optimum activity (1). Above 280 nm the enzyme
has absorption maxima at 337 nm and 420 nm whose relative
amplitudes depend on both pH and monovalent cations (2) .
Equilibrium studies have shown that the apparent pKa (pKa')
for the interconversion Of these spectral forms varies with
the nature and concentration of the monovalent cation and
that this pKa' is related to the Vmax for the breakdown of
the artificial substrate S-orthonitrOphenyl-L-cysteine (3).

We reported previously (4) that the 420 nm and 337 nm
forms of the enzyme interconvert in a complex fashion on the
scanning stOpped flow time scale following a rapid change in
pH or monovalent cation. This Observation provided us with
a unique Opportunity to study the spectral forms of trypto-
phanase in the absence of substrates or inhibitors.

The above-mentioned scanning stOpped flow studies have
been extended to include incremental pH jumps and drOps over
the range Of enzyme stability. Major features of the data
are interpreted in terms Of a simple model including kinetic

constants and postulated structures.

55

MATERIALS AND METHODS

 

Materials - KCl was Mallinckrodt, analytical reagent.

 

2(N-morpholino) ethane sulfonic acid (Mes), N-tris(hydroxy-
methyl) methyl-Z-aminoethane sulfonic acid (Tes), N-Z-
hydroxyethylpiperazine prOpane sulfonic acid (Epps),
N,N-bis (Z-hydroxyethyl) glycine (Bicine), pyridoxal-5‘-
phOSphate, and DL-dithiothreitol (DTT) were Obtained from
Sigma Chemical CO.

TryptOphanase - TryptOphanase was prepared according

 

to the method of Suelter et a2. (16). Stock apoenzyme was
activated by incubation for 1 hr. at 37°C in 0.1 M
potassium phosphate, pH 8.0, 1 mM EDTA, 7% (NH.);SO., 0.2 mM
pyridoxal-P and 20 mM DTT. The enzyme had a specific acti-
vity of 50 - 55 umol min"mg"when assayed at 30° C with

0.6 mM S-OrthonitrOphenyl-L-cysteine (SOPC) in 50 mM
potassium phOSphate, pH 8.0, and 50 mM KCl (3). Protein
concentration was determined Spectrophotometrically using

’ cm" (5).

8210' 0.795 ml mg-

Incremental pngump experiments - Activated holotrypto-

 

phanase (~20 mg ml'1 ) was equilibrated with 1 mM Tes, pH
7.00, 0.2 M KCl, 15 NM pyridoxal-P, 1 mM EDTA and 0.2 mM

DTT by dialysis at 4° C. Prior to use, the enzyme was
diluted to a concentration Of 2.9 mg ml“ with the dialysis
buffer. This solution was pushed against 50 mM Bicine, pHi,
0.2 M KCl, 15 uM pyridoxal-P, 1 mM EDTA to give the final pH
values of 7.69, 8.07, 8.56, 8.90 and 9.33.

56

Incremental pH drOp experiments - Activated holotrypto-
phanase (~20 mg ml'1 ) was equilibrated with 1 mM Bicine,
pH 8.70, 0.2 M KCl, 15 uM pyridoxal-P, 1 mM EDTA and 0.2 mM
DTT by dialysis at 4° C. The enzyme was diluted to a concen-
tration of 4.0 mg ml"prior to the experiment with the dialy-
sis buffer. This solution was pushed against 50 mM Tes, pHi,
0.2 M KCl, 15 pH pyridoxal-P, 1 mM EDTA to give final pH
values Of 6.54, 7.03, 7.47, 7.77, 8.25 and 8.44.

ScanningStOpped Flow Kinetics - StOpped flow data were

 

collected on a scanning instrument described elsewhere (6,7)
at 24.0 1 0.1° C. 150 spectra per second were collected
over the wavelength range 280 nm - 550 nm for a period of
65 seconds in the pH jump experiments and 111 seconds in the
pH drop experiments. A total of 50 and 56 spectra were
stored in the pH jump and drop experiments, respectively,
using an averaging scheme outlined previously (8). Control
Spectra were collected as described previously (4).

Data Analysis - Data were fitted to the apprOpriate
mathematical function using the nonlinear curve-fitting
program KINFIT (9). Errors listed are marginal standard
deviations. All titration data were fitted to equation 1

previously given by Johnson and Metzler (10).

Aobs . AA(10(pH'pK)) + AHA
(1)

 

(1 . IoCPH-PK))

57

where Aobs is equal to the Observed change in absorbance at
a given pH, AA is the calculated absorbance change at infin-
ite pH, AHA is the calculated change in absorbance at zero pH
and pK is the negative log Of the dissociation constant for
the monoprotic acid.

pH Measurements - pH values were determined with a

Beckman Model 4500 digital pH meter at 24° C.

RESULTS

As pointed out earlier, Marina and Snell (2) originally
showed that the 420 nm form of tryptOphanase predominates at
low pH values whereas at high pH values the 337 nm absorp-
tion predominates. The absorption spectra of tryptOphanase
at pH 7.00 and 8.70 obtained by scanning SpectroscOpy clearly
confirm these results (Figure 1A). TO investigate the time
dependent interconversion of these Spectral forms, the pH
Of an enzyme solution was increased (jumped) or decreased
(dropped) in a scanning stopped flow spectrOphotometer and
the absorbance was scanned as a function Of wavelength and
time as outlined in Materials and Methods. Typical results
Of a pH drOp experiment which are given in Figure 18 as
a 3-dimensional difference absorbance-wavelength-time surface
Show the region of the spectrum where changes occur. These
Spectral changes were analyzed in terms of 3 distinct

phases: 1) an abrupt phase, which is complete in less than

58

Figure l: Absorbance Spectrum of tryptOphanase at
low and high pH values and absorbance difference-wavelength-
time surface observed in a pH drOp experiment. A) Absorp-
tion Spectrum of tryptOphanase at pH 7.00 (O) and pH 8.70
(A); B) Absorbance difference-wavelength-time surface
following a drOp in pH from 8.70 to 6.54.

S9

 

0.20

.15
l

0

.10

l

HBSPRBRNCE

0.05

.00

 

 

 

 

 

350 400 450
NRVELENGTH [NM]

FIGURE 1A

60

FIGURE 1B

-0.06
-O.I2

A ABSORBANCE

61

5 msec, 2) a fast first order interconversion of 420 nm and
337 nm absorbance, and 3) a slow first order process invol-
ving growth at 355 nm coupled to two decays centered at

325 nm and 430 nm in the incremental pH jumps; and decay at
355 nm with concomitant growth at 430 nm and slight growth
at 290 nm in the case of the incremental pH drOp experiments.
Each Of these phases will be discussed separately.

Abrupt Spectral Changes - The First Phase - The differ-

 

ence spectra obtained by subtracting the enzyme Spectrum at
t . 0, prior to mixing, from the first Spectrum after mixing
(6 - 12 msec) are shown in Figure 2. These changes occurred
during the 6.5 msec dead time (1.85 cm-path-length cell) and
are referred to as the abrupt changes. A and B of Figure 2
show the difference Spectra Obtained in the incremental pH
jump experiments. The overall features of the Spectra
include a peak at ~295 nm, a generalized increase in absor-
bance in the wavelength range ~330 nm - 420 nm, with possible
peaks at ...340 nm and at ...395 nm, and a decrease in the range
~430 nm - 460 nm. C and D Of Figure 2 are abrupt differ-
ence spectra Obtained in the pH drOp experiments. These
spectra exhibit increases in absorbance centered at ~330 nm

and ~420 nm and a decrease at ~295 nm.

The complexity Of these difference spectra suggests
that several processes, perhaps involving rapid subtle

conformational changes and titration Of various functional

62

F1 ure 2: Abrupt difference Spectra obtained in the
incrementaI pH jump and drop experiments. A) Incremental

pH jumps from pH 7.00; pH 9.33 (0), pH 8.90 (A), pH 8.56 (+);
B) Incremental pH jumps from pH 7.00; pH 8.07 (0), pH 7.69
(A); C) Incremental pH drOps from pH 8.70; pH 6.54 (0),

pH 7.03 (A), pH 7.47 (+); D) Incremental pH drOps from pH

3.70; pH 7.77 (0), pH 8.25 (A), pH 8.44 (+).

63

P

<~ meanwu

fizzy Ihozmmm>¢z
mm. mam m m

r

mfim

ms

 

 

 

20°05“

10'0-

SO'

L0'0

 

 

 

64

EN otsuma

fizzy Ipozm4m>¢z

 

 

 

 

 

mmm mam mb¢ mmo mmm mmm mfim th_
. . _ r _ p p 0
me
I. 7...
3
l 2 ’ In—U
: . ..nlv
- . I 0%
. . OS
.4 \ . 00
, HO
.1 \ . 8
. ...: .. “U
... ‘- . \..\ x N
I. I I . .....H‘ .. :5 . Tmua
I .1 _ m3
1 3 2 0
J.
.. z
1 0
a A . a A . q mu
8

65

UN oasumu

 

 

 

 

 

 

 

 

fizzy Ipczw4m>¢3
www mam who mMV mmm mmm N.
. p r p p p nIU-
m5
L
1 lm
mu
.7
_ .IIU
. 7.5%
so \. LI... 00
.... 8
I as uufio
. . mad
3
I E. ..0
.. mu
7.. S
10
_ q q a a d mu
8

66

o~ «tamed

fizzy :Hozmmm>¢z
mmm mum we. mm. mam mmm mum ma

F _ p p P P

 

 

 

 

 

 

 

67

groups, contribute to alterations in the original Spectrum
that give rise to the difference Spectra. It is also likely
that some wavelength instability in the scanning stOpped
flow instrument itself contributed to the nonuniformity Of
these difference Spectra. Small wavelength shifts which
occurred during the time between the collection of the
various experimental spectra could cause wavelength shifts
in these difference spectra.

Although the difference Spectra for the pH jumps and
drOps are qualitatively different, the 295 nm peak which
appears in the pH jump difference spectra (Figure 2A, B)
occurs again, essentially as a mirror image, in the pH
drOp Spectra (Figure 2C, D). TO determine apparent pKa
values for the process at 295 nm in the pH jump and drOp
experiments, the change in absorbance at 295 nm was examined
as a function Of pH in each case (Figures 3A and 3B).

The data are not sufficient to clearly define pKa'
values in either case. Nevertheless, some qualitative
features can be discerned. It is clear, for example, that
the pKa' at 295 nm in the pH jumps is above 9.0. To illus-
trate this the theoretical line was drawn with a pKa' value
of 9.6 t 1.2, Obtained by fitting the data to equation 1
with KINFIT (9). The data for the pH drOp experiments,

which extend the lower range Of pH investigated to 6.54,

68

The change in absorbance at 295 nm as a

Figure 3.
funct1on O pH for the abrupt spectral changes Observed
in the incremental pH jump and drop experiments. A) Incre-

mental pH jumps. The line was calculated using equation 1
with a pK value of 9.63. B) Incremental pH drOpS. The
line was calculated with pK values Of 6.3 and 9.4.

69

 

0.20

295NM]
15

RNCE I
0 0

-1

880
0

0:05

DELTR R

.00

l

 

.P

.00

0

 

 

8100

I

g-o
0-0
D
O

9100 10.00
PH

FIGURE 3A

70

 

 

7.50 8.50 9.50

PH

6.50

 

o~.o

mo. 01 o”. DI
8n.zzmmN.o .mozmmmowmc mhqwo

 

5.50

m~.cl

33

FIGURE

71

indicate that two groups may be involved when the pH is
drOpped incrementally from a starting pH of 8.70. The
theoretical line was drawn with pKa,‘ = 6.3 and pKaz' =
9.4 t 4.7.

Fast First Order Interconversion of 420 nm and 337 nm

Forms of Tryptophanase - The Second Phase - The second phase,

 

which occurs after a change in pH, is a fast first order
interconversion Of 420 nm and 337 nm absorption. A and B

Of Figure 4 Show the spectral changes Observed as a function
Of time following an increase in pH from 7.00 to 9.33 and a
decrease in pH from 8.70 to 6.54, reSpectively. After such
a pH jump, there is a growth centered at ~337 nm coupled to
decays at ~420 nm and ~290 nm. The changes Observed after

a decrease in pH mirror those seen after a pH increase.
Isosbestic points occur at ~300 nm and ~360 nm in both
cases.

The first order rate constants for the changes at 420 nm
and 337 nm were Obtained by fitting the absorbance vs. time
data at each wavelength to equation 2 with KINFIT (9). The
adjustable parameters were Am, the absorbance at infinite
time, AA, the

Aobs - Ana 1 AAe(-k1't) (2)
change in absorbance [i.e., (Am - A0), where A0 is the
absorbance at t s 0], and k-‘, the apparent first order rate

constant .

72

Figure 4. Spectral changes occurring during the fast
first order interconversion of 420 nm and 337 nm absorptions.
These difference spectra were Obtained by subtracting the
spectrum collected before any changes occurred from the
spectrum collected after the fast first order changes were
complete. A) A pH jump from pH 7.00 to 9.33; B) A pH drop
from pH 8.70 to 6.54.

73

 

.18

0.08
L

 

-0 002

I

 

I

DELTH RBSORBHNCE

-0.12

 

 

 

 

01
OH
C)

N,-0.22

75 350 355 420 455
NHVELENGTH (NM)

FIGURE 4A

74

 

 

 

 

 

I
550

 

o._o
wuzcmm wmq a

5
l9
4
0H
I4N
4[
H
T
nu
SN
E
l8
3L
E
V
H
N
0
[3
3
5
7
2
m~.oI

 

FIGURE 4B

75

Apparent pKa values for this interconversion process
were Obtained by fitting the absolute values of AA, Obtained
as described above, at 337 nm and 420 nm as a function Of
the final pH to equation 1 as a multiple data set (Figures
5A and 5B). This gave pKa' values, respectively, of
8.30 t 0.02 and 8.08 t 0.04 for the pH jump and drOp
experiments.

The first order rate constants for the changes in absor-
bance at 337 nm and 420 nm Obtained in each pH jump or drOp
experiment were essentially identical. However, they varied
with pH in a systematic manner as indicated in Table I and
Figure 9. The data for the change centered at 290 nm
appeared to Show the same kinetic behavior as the 420 nm and
337 nm data. However, the noise encountered at this low
wavelength coupled with small absorbance changes, especially
in the smaller increments of pH change, made analysis diffi-
cult. Values are reported in Table I for those instances
where the analysis was successful.

Representative plots showing the changes occurring at
295 nm, 337 nm and 420 nm as a function of time for a pH
jump from 7.00 to 9.33 are shown in Figures 6A, B and C.

The theoretical lines were calculated with equation 2

using the parameters listed in Table I.

76

Fi ure 5. The changes in absorbance at 337 nm and
420 nm oEserved in the incremental pH jump and drOp
experiments as a function Of pH. A) Incremental pH
jumps; 337 nm (0), 420 nm (A). The lines were calculated
using equation 1 with a pK value of 8.30; B) Incremental
pH drOps; 337 nm (0), 420 nm (A). The lines were calculated
using equation 1 with a pK value of 8.08.

77

 

0.21

LLJ<r
L3":

.07

AORBSORBFI

 

.00

 

 

C’7.00 8100 9100 10.00
PH

FIGURE 5A

78

 

 

7100

 

 

mn.o

JI

m~.o

pace
muzcmmowmm

"one
q

wo.o

6.00

FIGURE SB

Table I:

79

Kinetic parameters for the fast first order
interconversion Of 420 nm and 337 nm absorbance
in the pH jump and drOp experiments. These
parameters were Obtained by fitting the changes
in absorbance at each wavelength to equation 2.

INCREMENTAL pH JUMP EXPERIMENTS:

 

 

 

 

- 1 five-
F138 length .Am AA k,‘ (sec")
p (nm)

9.33 295 0.711 1 0.007 0.0674 1 0.0020 1.79 1 0.38
337 0.325 1 0.003 0.154 1 0.003 2.78 1 0.08
420 0.169 1 0.003 0.206 1 0.003 2.63 1 0.01

8.90 295 0.687 1 0.008 0.0553 1 0.0260 1.06 1 0.35
337 0.229 1 0.003 0.133 1 0.002 1.44 1 0.01
420 0.207 1 0.002 0.182 1 0.003 1.38 1 0.04

8.56 295 0.687 1 0.005 0.0362 1 0.0035 0.620 1 0.156
337 0.273 1 0.002 0.106 1 0.002 0.882 1 0.004
420 0.248 1 0.002 0.146 1 0.001 0.838 1 0.004

8.07 337 0.236 1 0.002 0.0630 1 0.0005 0.464 1 0.015
420 0.310 1 0.004 0.0863 1 0.0013 0.423 1 0.007

7.69 337 0.213 1 0.001 0.0334 1 0.0004 0.359 1 0.006
420 0.353 1 0.004 0.0466 1 0.0007 0.338 1 0.004

INCREMENTAL pH DROP EXPERIMENTS:

6.54 295 0.879 1 0.019 0.070 1 0.012 0.483 1 0.016
337 0.186 t 0.002 0.138 t 0.001 0.541 t 0.010
420 0.329 1 0.003 0.181 t 0.003 0.519 t 0.004

7.03 295 0.889 1 0.005 0.048 1 0.001 0.487 1 0.123
337 0.207 1 0.003 0.127 1 0.001 0.414 t 0.002
420 0.319 t 0.003 0.169 t 0.001 0.409 t 0.003

7.47 337 0.236 1 0.001 0.104 t 0.001 0.366 t 0.005
420 0.296 1 0.001 0.140 1 0.001 0.362 1 0.004

7.77 337 0.258 t 0.001 0.0818 1 0.0010 0.384 t 0.003
420 0.264 1 0.001 0.109 1 0.001 0.377 1 0.007

8.25 337 0.304 1 0.001 0.0377 1 0.0011 0.534 1 0.032
420 0.209 1 0.001 0.0498 1 0.0005 0.507 1 0.009

8.40 337 0.323 1 0.002 0.0205 1 0.0006 0.665 1 0.118
420 0.186 t 0.002 0.0262 1 0.0005 0.620 1 0.053

 

80

Fi ure 6. Changes in absorbance at 295 nm, 337 nm and
420 nm as a function of time during the fast first order
conversion of 420 nm to 337 nm absorbance following a jump
in pH from 7.00 to 9.33. A) first order decay of 295 nm,
B) first order growth at 337 nm, C) first order decay at
420 nm. The lines through the data were calculated from
equation 2 and the parameters listed in Table I.

81

 

1.50 2.00

I

Loo
TIME (SEC)

0.50

 

 

 

0
AIDIQ 0 - P . q
bh.o mh.o _ Nb.o
522mmmu muzcmmommc

 

9100

05.

FIGURE 6A

RBSURBRNCE (337NH)

82

 

0.35
%

    

0.30
g

l

0.25

 

 

 

ch'.00 0'.50 1300 1'.50 3500
TIME (SEC)

FIGURE 68

 

 

mm.o

1.50 2.00

1
1.00

TIME (SEC)

0.50

 

mmuo swan fiuuo
nzzowvu wuzcmmowmc

mu.

 

c0.00

FIGURE 6C

84

Slow First Order Processes - The Third Phase - Incremen-

 

talng Jump Experiments - Figure 7A shows the time dependence

of the changes in Spectra which occur in the so-called slow

first order process following a sudden increase in pH from

7.00 to 9.33. These changes involve growth at ~355 nm with

concomitant decays centered at ~325 nm and ~430 nm. Similar

changes were observed at the four other final pH values. The

absorbance 06. time data at the above three wavelengths were

fitted to equation 2 to obtain values for Aw, AA and kz',

the apparent first order rate constant for the slow phase.
The rate of the slow third phase, kz', was independent

of wavelength and pH and gave an average value of 0.064 1

0.005 sec'1 for five determinations at 355 nm and 420 nm.

The data at ~325 nm were somewhat noisier than data collected

at higher wavelengths and consequently the analysis was less

successful at this wavelength. Table 11 lists the values

for An, AA and k2' for the incremental pH jumps. Analysis

of the change in absorbance at 355 nm as a function of pH

using equation 1 in KINFIT yielded a pKa' value of 8.10 1

0.06 (Figure 8). Thus, this slow process exhibits essentially

the same pH dependence as the fast interconversion of the

420 nm and 337 nm forms of the enzyme in the pH jump and

drop experiments.

85

Fi ure 7. Spectral changes occurring during the slow
first order process following a rapid change in pH. These
difference spectra were obtained by subtracting the spectrum
collected after the fast first order changes were essentially
complete, but before the slow first order process had begun,
from the last spectrum collected. A) A pH jump from 7.00
to 9.33; B) A pH drop from 8.70 to 6.54.

 

86

 

0.08

.05
J

U

.02

I

9%80RBRNCE

 

l

DELTH
-0.01

   

 

 

 

N5-U-04

75 350 305 420 405 550
NRVELENGTH (NM)

FIGURE 7A

0.05

.03
1

0

.01
1

9%SORBRNCE

DELTR
-U.01

l

87

 

‘ ’ V \

“\
It I
”In
.1. .... ‘
. . :3”)?

- mun.
'0‘ "w
‘Vw ...

 

 

 

 

“;0.03

 

75

350 305 420 455 50
NRVELENGTH (NM)

FIGURE 7B

 

88

 

 

Table II: Kinetic parameters for the slow first order
process in the incremental pH jump experiments.
These parameters were obtained by fitting the
changes in absorbance at each wavelength to
equation 2.
Final Wave-
pH length Aw AA kz' (sec")
(nm)
9.33 325 0.279 1 0.002 0.040 1 0.003 0.042 1 0.005
355 0.270 1 0.003 0.082 1 0.001 0.058 1 0.002
430 0.146 1 0.003 0.024 1 0.001 0.068 1 0.013
8.90 325 0.268 1 0.003 0.025 1 0.004 0.043 1 0.008
355 0.265 1 0.004 0.076 1 0.001 0.062 1 0.003
430 0.174 1 0.002 0.039 1 0.002 0.068 1 0.006
8.56 325 0.254 1 0.003 0.013 1 0.003 0.029 1 0.015
355 0.252 1 0.002 0.065 1 0.001 0.065 1 0.002
430 0.213 1 0.002 0.048 1 0.001 0.066 1 0.001
8.07 355 0.225 1 0.003 0.037 1 0.001 0.062 1 0.002
430 0.287 1 0.005 0.040 1 0.005 0.062 1 0.009
7.69 355 0.207 1 0.001 0.018 1 0.001 0.073 1 0.006
430 0.340 1 0.003 0.023 1 0.001 0.060 1 0.009

 

89

F1 ure 8. The change in absorbance at 355 nm as a
function of pH observed in the slow first order process
in the incremental pH jump experiments. The line was
calculated using equation 1 with a pK value of 8.10.

 

90

10

 

 

 

 

7.00 8'.00
PH

FIGURE 8

9

.00

10.00

91

Incremental pH DrOp Experiments - Changes in Spectra

 

which occur during the slow first order process following a
dr0p in pH from 8.70 to 6.54 (Figure 7B) involve a decay at
~355 nm and growths at ~430 nm and ~290 nm. These changes
are essentially the same as those observed at the other five
final pH values. The growth at 290 nm, however, becomes
negligible as the extent of the pH drop becomes smaller.

The values of k,’, for the pH drop from 8.70 to 6.54
were 0.009 1 0.004 sec'1 and 0.008 1 0.001 sec"1 at 355 nm
and 430 nm respectively. The rate constants are just esti-
mates since the data were collected for less than two half
times. This made it impossible to obtain a reliable value
of Aon so that the apparent pKa for the conversion of the
355 nm absorbance to that at 430 nm could not be obtained.

Interpretation of Experimental Results and Discussion -
The experimental data presented above were analyzed in terms
of the kinetic model outlined in Scheme 1. This scheme
assumes that the enzyme can exist in at least three confor-
mations and that interconversion between these forms is a
relatively slow process on the stOpped flow time scale. In
addition, each of these enzyme conformations has associated
with it forms of pyridoxal-P which exhibit different absorp-

tion maxima. For example, conformation 8 contains forms of

92

m min—Sm

 

 

 

 

 

ZOfigae—‘OU

93

the coenzyme which absorb at ~420 nm while conformations

y and 6 have forms that absorb at ~337 nm and ~360 nm,
respectively. The structures giving rise to these various
absorptions are known from both model studies and studies
on other pyridoxal-P dependent enzymes (10).

In two of the three conformations, namely 8 and y, the
forms of the coenzyme within each conformational manifold
appear to differ only by their state of protonation at
the pyridinium nitrogen. Protonation at this site does
not greatly alter the location of the absorption maxima of
pyridoxal-P Schiff's bases although the extinction
coefficient may vary somewhat (10). Thus both HIEB and 138
would be eXpected to show an absorption maximum at ~420 nm,
while the HIEY and EY absorption would be centered at
~337 nm. E5, which absorbs at ~355 nm, appears to be
essentially the form of the coenzyme which is associated
with activity in aSpartate aminotransferase (11). This is
also approximately the wavelength associated with the maximal
rate of photoinactivation of tryptOphanase, (12) and thus
E5 may play a role in this process.

The abrumpt changes presumably reflect protonations
or deprotonations occurring within conformation manifolds.
According to Scheme I these abrupt changes would involve
protonation or deprotonation at the pyridinium nitrogen.

The fast and slow first order processes would then reflect

94

conversions between conformations. The fast process involves
the 420 nm and 337 nm absorption bands and reflects conver-
sions between conformations B and y. The slow process which
shows absorption changes at ~325 nm, ~355 nm and ~430 nm

in the pH jumps and at ~355 nm and ~430 nm in the pH draps
involves all three conformations.

If we assume that the fast first order process involves
conformations 8 and y as well as the four structures of
pyridoxal-P therein, we can obtain an expression for the
apparent first order rate of appearance or disappearance
of the 420 nm or 337 nm absorbance (see Appendix A, part A,

for derivation):

(k-,K./[H*] +k,) + (K,/K.)(k,/k-,)k, + (k,K,/[H*1) (3)
k1' ' (1 + K~/[H*l) (1 + x./[n+]

 

 

The rate and equilibrium constants are defined in Scheme I.
The parameters obtained from a fit to equation 3 of the

apparent first order rate constants at 337 nm and 420 nm as
a function of pH for both the pH jump and dr0p experiments
are pKa = 9.5 1 0.1, pK. - 6.2 1 0.3, k; = 6.3 1 0.9 sec",

k_, - 0.23 1 0.02 sec", k, - 1.1 1 0.3 sec", k - 0.015 1

'3
0.004 sec". The theoretical curve through the experimental
values for k,’ in Figure 9 was drawn using these parameters.
If we ignore conformation 6, which is always formed to a

small extent even at high pH values, the percentage of

95

Fi ure 9. Variation of the apparent first order rate
constant for the fast first order interconversion of 420 nm
and 337 nm absorptions in the incremental pH jump and drOp
experiments. The apparent first order rate constant was
determined at 337 nm and 420 nm for each individual ush.
Each symbol on the graph represents the average of t ree
pushes. Incremental pH jump experiments; kz' from 337 nm
data (0), k,’ from 420 nm data (A). Incremental pH drop
experiments; kx' from 337 nm data (+), k1' from 420 nm
data (0). The line was calculated using equation 4 with
the parameters listed in the text.

 

96

 

 

 

 

C3
C? 1 l 1
07-
CD
r—\CD
ESE UN" ’
hm LLJ
iS (0
5h. \
38 H
n? ~—ICD
up CD
:h vdrz- —
:C
CD
CD
C., l I I
6.50 7.50 8.50 9.50

PH

Figure 9

97

enzyme in each of the forms HIEB , EB, HIEY and BY can be
calculated with the parameters obtained with equation 3.
Table III which lists the percentages of these four species
at three pH values reveals that EB and HIEY are always
relatively minor components. HIEB and EY make the largest
contribution to the absorbance at 420 nm and 337 nm, respec-
tively. The distribution between these two absorption bands
at a given pH value depends on the effective pKa,
p[Ko(k1/k_;)] . 8.1. This effective pKa is in close
agreement with the value of 8.3 obtained by equilibrium
titrations for the pH dependent interconversion of the

420 nm to 337 nm absorption in 0.2 M KCl. This close
agreement between equilibrium and kinetic results supports
our contention that at any given pH most of the enzyme is

in the forms H‘E and By. Thus, changes in these forms

8
dominate the equilibrium spectrum.

 

Table III: The percentages of total enzyme that exists
in each of the four forms HIEB , EB, H+EY
and BY at three pH values.

 

+ +
pH 15 BB 153 an BY 15,
7.00 91. .27 1.2 7.3
3.00 54. 1.6 .72 44.

9.00 11. 3.2 .14 86.

 

98

As mentioned previously, the abrupt Spectral changes
observed in the pH jump and drop experiments (Figure 2) are
complex and cannot be interpreted in terms of one or two
ionizing groups. However, it is interesting to note that
the titration curves obtained by plotting the change in
absorbance at 295 nm 06. pH for the pH jumps (Figure 3A)
and the pH drOpS (Figure 3B) reveal pKa' values of approx-
imately 9.6 and 6.3, respectively. There is also an indi-
cation of a higher pKa of uncertain value in the pH drOp
experiments (Figure 3B). Although these pKa’ values are
not well determined, the values of 9.6 and 6.3 are consistent
with the values obtained by analysis of the more reliable
kinetic data to give 9.5 and 6.2 for pKo and pK,, reSpec-
tively. Thus, the change in absorption at 295 nm, as well
as other features in the abrupt difference spectra may
arise as a result of protonation of EY in the case of a

pH drOp or deprotonation of H‘E in the case of a pH jump,

B
at the pyridinium nitrogen.
Figure 10 shows the absorption spectra of the pyridoxal-
P valine Schiff's base forms HzPL, HPL, and PL which corres-
pond to “+EB’ EY and E6 respectively. H2PL and HPL have
similar Spectra with peaks at ~413 nm and ~280 nm.
Figure 11 is the difference spectrum that results when

the H2PL Spectrum is subtracted from the HPL spectrum. This

corresponds to the difference Spectrum obtained during the

99

Fi ure 10. Absorption Spectra of various ionic
forms oi pyridoxal-P. HzPL (0), HPL (A), PL (1). Spectra

were kindly supplied by Prof. D. E. Metzler, Iowa State
University.

100

o~ masoum

mmmzazméz
8.2. 86% 8.3m 861,0. -85

      

00-0N

2-0V 31303 NOIlONIlXB 55100

.....
A D . .
.......

1

00'08

00'09

 

 

 

00'06

101

Fi ure 11. Difference Spectrum obtained by sub-
tracting the HzPL spectrum from the HPL spectrum, both

given in Figure 10.

120.00

11(1‘

80.00

40.00

I

DELTR 9%SURBRNCE

‘40-00

Nr80.00

102

 

l

.00

  
  

 

 

l

   

 

 

 

50

300

350 400 450 500
NRVELENGTH (NM)

FIGURE 11

103

during the abrupt changes of a pH jump with tryptOphanase.
A comparison of Figure 11 with Figures 2A and B reveals
definite similarities in spectral shapes in support of

our argument that deprotonation at the ring nitrogen con-
tributes to these difference spectra. Figures 2C and D

are essentially the reverse of Figure 11 in the sense that
these would represent a protonation at the pyridinium nitro-
gen. The discrepancies between wavelength maxima for
difference peaks and valleys between Figures 2 and 11 may
arise as a result of the differing environments of the
pyridoxal-P imine in tryptophanase and the model compound.
The changes at 295 nm, on the other hand, could reflect a
rapid change in the environment of one or more enzyme
tryptophan residues Since this is the wavelength of maximum
difference for tryptOphan perturbations (13). This inter-
pretation would be consistent with the results of Tokushige
et a2. (14) who suggest that there is a tryptOphyl residue
at the active Site of tryptophanase in close proximity to
the coenzyme moiety, which is necessary for catalytic
activity and pyridoxal-P binding. This tryptophyl residue
may be very sensitive to Slight perturbations caused by
changes in the conformation or the change distribution of
the bound coenzyme. Regardless of which mechanism pertains,
the change centered at 295 nm is the most constant feature

in the abrupt difference spectra for the pH jumps and draps

104

and appears to reflect the pKa values obtained in the
kinetic analysis. Spectral changes at 295 nm may be the
most sensitive probe for the state of protonation at the
pyridinium nitrOgen of the bound pyridoxal-P of trypto-
phanase.

The addition of the conformation manifold 6, containing
the species Ed serves to rationalize the changes seen in
the incremental pH jump and drOp experiments during the slow
first order process. For example, in the pH jump experiments
there is an increase in absorbance at approximately 355 nm
correSponding to the formation of E5. The decreases in
absorbance at ~430 nm and ~325 nm which occur at the same
rate as the appearance of 355 nm absorbance correspond to
the conversion of HIEB and By, respectively, into B5. The
rate constants obtained for the interconversion of the B
and Y manifolds Show that these steps are fast enough to
remain at equilibrium during the formation of the 6 confor-
mation. This is the explanation of why the first order
rates are the same at these three wavelengths.

In the case of the pH drOp experiments there was a
decrease in absorbance at ~355 nm with a concomitant
increase at ~430 nm. These changes would correspond to
the disappearance of E6 and the appearance of H‘BB,

respectively. Absorbance at ~325 nm would not be expected

105

to build up according to the proposed mechanism because as
soon as EY is formed from E5 it rapidly forms HIEB at a rate
which is much faster than the rate of conversion of E5 to
By.

A derivation (see Appendix A, part B) of the rate of
change of E5 as a function of time for the above mechanism
gives the following equation for the apparent first order
rate constant for the appearance or disappearance of E5 at

any final pH:

k2' = k2 * k-2

(1 * K1 + [H+]/K0K1 * IH+I/K0K1K3)
Although the value of K2, the equilibrium constant for the
formation of E6 from EY’ is not known, it must be less than
one because B5 is not formed to any great extent even at
pH 9.33 where most of the enzyme is in the form of By. Since
K: = k2/k_2, this implies that k2 is then less than k_2,
Substituting the known values of K., K; and K, of 2.95E-10M,
27.2 and 73.3 respectively, into equation 4 reveals that
the contribution of the terms (H*)/K.K1and (H+)/K.K1K, in
the denominator, through which any pH dependence is expressed,
is small at all pH values studied in the pH jump experiments
compared to 28.2, the sum of l + K1. This, coupled to the
fact that k2 is small in comparison to k_2 gives rise to the
observed pH independence of k2' as a function of pH in the

incremental pH jump experiments.

106

It should be pointed out that this derivation does not
explain the 7.5-fold difference between kz' observed in the
pH jump and drOp experiments. In a somewhat related study
on the pyridoxal-P dependent enzyme glutamate decarboxylase
(15) O'Leary and Brummund observed that the rate of conver-
sion of a Species absorbing at 420 nm following a rapid
decrease in pH was affected by the nature and concentration
of the buffer used. It is conceivable that the rate of
interconversion of the y and 6 conformation manifolds is
also sensitive to buffer effects. Additional experimentation
under various buffer conditions will be required to resolve
this discrepancy.

The increase in absorbance at ~295 nm during the Slow
phase at the lowest pH values may be indicative of yet
another group on the enzyme with a very low pKa' or further
perturbation of tryptOphan residues. Essentially no infor-
mation is available about this process, however, due to

enzyme instability at low pH values.

10.

ll.

12.

13.

I4.

107

REFERENCES

Snell, E. E., (1975) Adv. Enzymol. Relat. Areas Mol.
Biol. 42, 287-333.

 

Morino, Y., and Snell, E. E. (1967) J. Biol. Chem.
242, 5591-5601.

Suelter, C. H., Wang, J., and Snell, E. E. (1976)
FEBS Lett. 66, 230-232.

 

June, D. S., Kennedy, B., Pierce, T. H., Elias, S. V.,
Halaka, F., Behbahani, Nejad, 1., El-Bayoumi, A.,
Suelter, C. H., and Dye, J. L., (1979) J. Amer. Chem.
§gg.‘lgl, 2218-2219.

H6gberg-Raibaud, A., Raibaud, 0., and Goldberg, M. E.
(1975) J. Biol. Chem. 250, 3352-3358.

 

Coolen, R. B., Papadakis, N., Avery, J., Enke, C. G.,
and Dye, J. L. (1975) Anal. Chem. 41, 1649-1655.

Papadakis, N., Coolen, R. B., and Dye, J. L. (1975)
Anal. Chem. 41, 1644-1649.

Suelter, C. H., Coolen, R. B. and Dye, J. L. (1975)
Anal. Biochem. 62, 155-163.

 

Dye, J. L., and Nicely, V. A. (1971) J. Chem. Ed. 18,
443-448.

Johnson, R. J., and Metzler, D. E. (1970) Methods
Enzymol. 18A, 433-471.

Ivanov, V. I., and Karpeisky, M. Ya. (1969) Adv.
Enzymol. Relat. Areas Mol. Biol. 32, 21-53.

 

Coetzee, W. F., and Pollard, E. C. (1975) Photochem.
and Photobiol. 22, 29-32.

Kayne, F. J., and Suelter, C. H. (1965) J. Amer. Chem.
Soc. 81, 987-900.

Tokushige, M., Fukada, Y., and Watanabe, Y. (1979)
Biochem. BiOphys. Res. Comm. 86, 976-981.

 

108

References - continued
15. O'Leary, M. H., and Brummund, W. (1974) J. Biol. Chem.
249, 3737-3745.

16. Suelter, C. H., Wang, J., and Snell, E. E. (1977)
Anal. Biochem. 16, 221-232.

 

SECTION IV
SCANNING STOPPED FLOW STUDIES ON THE MECHANISM OF QUINONOID
FORMATION WITH TRYPTOPHANASE USING
COMPETITIVE INHIBITORS

109

An absorption band centered around 500 nm appears
following addition of certain amino acid inhibitors to
several pyridoxal-P dependent enzymes (1 - 6). This band
has been attributed to a quinonoid complex at the enzyme
active site formed by the loss of a leaving group from the
o-carbon of the amino acid-pyridoxal-P Schiff's base (7).
Morino and Snell (3) demonstrated that a Stable, dead-end
quinoid complex, characterized by an intense absorption
band centered at 502 nm was produced when the competitive
inhibitor, L-alanine, interacted with tryptophanase and
that the o-proton of alanine was labilized in the process.
Watanabe and Snell (8) later identified a number of inhib-
itors that formed quinonoid derivatives with tryptOphanase.

The formation of the tryptOphanase-quinonoid complex
with ethionine has an absolute requirement for monovalent
cations (9). Suelter and Snell showed that in the absence
of cations, ethionine interacted with the enzyme, presumably
forming a Schiff's base with coenzyme, but was unable to
proceed to quinonoid until KCl was added.

The purpose of this study was to examine the transient
kinetics of quinonoid production with inhibitors in an
attempt to gain insight into the mechanism of tryptOphanase
catalysis up to and including a-proton abstraction. Speci-

fically, this paper describes the results of stOpped flow

110

studies of the kinetics of formation of the quinonoid deri-
vative with L-ethionine and L-alanine. The effects of pH,
concentration of inhibitor, differing monovalent cations,
and the substitution of deuterium at the a-position of
alanine on this reaction are reported. These observations
are integrated with previous results into a simple mechan-

ism of tryptOphanase catalysis.
MATERIALS AND METHODS

Materials - KCl and NH,C1 were Mallinckrodt, analytical

 

reagents. CSCl and RbCl were obtained from Ventron, Danvers,
Mass. as ultrapure products. Pyridoxal-5'-phOSphate, L-
alanine, DL-dithiothreitol (DTT), and N-Z-hydroxyethyl-
piperazine prOpane sulfonic acid (Epps) were obtained from
Sigma Chemical Co. L-ethionine was from Vega Biochemicals
and L-[o-zHlalanine was purchased from Merck and Co., Inc.,
Rahway, N.J. (CH,),NC1 from Aldrich Chemical Co. was
recrystallized from isoprOpyl alcohol before use. (CH3).NOH
was prepared fresh before use by passage of recrystallized
(CH,),NC1 over Dowex-l—OH.

TryptOphanase - TryptOphanase was prepared according

 

to the method of Suelter et a2. (10). Stock apoenzyme was
activated by incubation for one hour at 37°C in 0.1 M

potassium phosphate, pH 8.0, 1 mM EDTA, 7% (NH.)zSO~,

111

0.2 mM pyridoxal-P and 20 mM DTT. Unless otherwise indi-
cated, the enzyme had a Specific activity of 50 - 55 umol
min'l mg"l when assayed at 30° C with 0.6 mM S-orthonitro-
phenyl-L-cysteine (SOPC) in 50 mM potassium phosphate,

pH 8.0, and 50 mM KCl (11). Protein concentration was
determined spectrOphotometrically using c - 0.795 ml mg"l
cm'1 (12).

Scanning StOpped Flow Experiments - These experiments
were completed with a computerized double beam rapid scanning
Stopped flow instrument described elsewhere (13, 14) at
24.0 1 0.1° C using a 1.85 cm path length. Scanning data
were collected using an averaging scheme previously des-
cribed (15) at a rate of 150 Spectra per second over the
wavelength range 280 nm - 600 nm. Control Spectra were
collected as outlined previously (16). Experimental
conditions for individual experiments are described below.

Tryptophanase Vb. L-ethionine and L-alanine - Activated
holotryptOphanase (~20 mg ml") was equilibrated by dialysis
at 4° C with 25 mM K-Epps, pH 8.0, 0.1 M KCl, 1 mM EDTA,

0.1 mM pyridoxal-P, 0.2 mM DTT and diluted to a concentra-
tion of 1.4 mg ml"1 with the same buffer. This enzyme
solution was pushed against various concentrations of
L-ethionine and L-alanine in 25 mM K-Epps, pH 8.0, 0.1 M
KCl.

112

For the data presented in Figures 1, 3 and 5 holotryp-
tOphanase was equilibrated with 25 mM K-Epps, pH 8.0, 0.2 M
KCl, 1 mM EDTA, 10 0M pyridoxal-P, 0.2 mM DTT and then
diluted to a concentration of 2.0 mg ml’1 before being
pushed against 20 mM L-ethionine in the same buffer.

TryptOphanase VA. L-(a-‘H1alanine and L-[a-zHJalanine -
Activated holotryptOphanase (~20 mg ml") was equilibrated
by dialysis at 4° C with 25 mM K-Epps, pH 8.0, 0.1 M KCl,

1 mM EDTA, 0.1 mM pyridoxal-P and 0.2 mM DTT and diluted to
a concentration of 1.8 mg ml". This enzyme solution was
pushed against 25 mM K-Epps, pH 8.0, 0.1 M KCl, 1 mM EDTA,
0.1 mM pyridoxal-P containing 0.45 M L-[a-’H]alanine or
L-[a-ZH]alanine.

Tryptophanase 06. L-ethionine at three pH values -
Activated holotryptOphanase (~20 mg ml") was equilibrated
by dialysis at 4° C with 25 mM K-Epps, pH 8.0, 0.1 M KCl,

1 mM EDTA, 0.1 mM pyridoxal-P and 0.2 mM DTT. The enzyme
was divided into three equal volumes at a concentration
of 1.6 mg ml". These three solutions were adjusted to
pH values of 7.2, 8.0 and 8.7, with 0.1 M HCl or 0.1 M
KOH at 24° C and pushed against 25 mM K-Epps, 0.1 M KCl
and 10 mM L-ethionine at a pH of 7.2, 8.0 or 8.7.

Tryptophanase-ethionine complex (minus cations) 06.
NH.Cl, KCl, RbCl and CsCl - Activated holotryptOphanase
(~25 mg ml") was dialyzed at 4° C against 25 mM (CH:)~N-
Epps, pH 8.0, 0.2 mM pyridoxal-P, 0.2 mM DTT and 1 mM

113

[(CH,),N)2-EDTA to remove activating cations. After this
treatment the enzyme had a specific activity of 30 umol
min" mg'.1 when assayed as described above with SOPC at
30° C.

The enzyme was diluted with the above buffer containing
10 mM L-ethionine to a concentration of 1.9 mg ml". This
solution was pushed against 25 mM (CH,).N-Epps, pH 8.0,
containing 0.05 M NH.C1, 0.2 M KCl, 0.5 M RbCl or 1.0 M
CsCl. No attempt was made to control ionic strength due
to the lack of a suitable non-activating salt [(CH,).NC1
is known to inhibit tryptOphanase at concentrations greater
than 50 mM (9)].

Data Analysis - Data were fitted to the apprOpriate
mathematical function using the nonlinear curve fitting
program KINFIT (l7). Errors listed are marginal standard
deviations.

All single exponential data were fitted to equation 1

At = A 1 AAe'k“t (1)

no

where A”, the absorbance at infinite time, AA, the total
change in absorbance [i.e. (Am-A0) where Ao equals the
absorbance at t - 0] and k,', the apparent first order

rate constant, were the three adjustable parameters.

114

Biphasic data were analyzed as the sum of two expon-

ential growths according to equation 2

A = Am - AA,(e'k1't)- AA,(e‘k2't) (2)

t

where A0° is the absorbance at infinite time, AA: and AA;
are the total changes in absorbance due to the fast and
slow phases, respectively, and k1' and k2' are the apparent
first order rate constants for the fast and slow phases.

pH Measurements - pH values were determined with a

Beckman Model 4500 digital pH meter at 24° C.

RESULTS

The first studies to be described were designed to
investigate the interaction of the dead-end inhibitors,
alanine and ethionine, with tryptOphanase on the StOpped
flow time scale. Digitized data, which were collected
with the rapid scanning stOpped flow device (l3, 14, 15)
and stored on floppy disk, were displayed on a cathode
ray tube for visual inSpection. Figure l is a photograph
of this diSplay showing a composite of 55 spectra collected
over a 65 second time period during the reaction between
tryptOphanase and ethionine. Spectra displayed in this
fashion (absorbance VA. wavelength) point out the exis-
tence of channels which are themselves composed of

absorbance 06. time data at a particular wavelength.

115

Tryptophanase Spectra collected as a

Fi ure 1.
function of time after mixing with ethionine at 24° C
in 25 mM K-Epps, pH 8.0, 0.2 M KCl, 1 mM EDTA, 10 0M
pyridoxal-P, 0.2 mM DTT. After mixing: 10 mM ethionine

and 1.0 mg ml"trypt0phanase.

116

H wmaumu

1E5 Ikozw4m><3
com on... com one 09. 0mm awn

 

 

 

 

 

_ p _ _ _ _ _ 00.0
........— ————-....._—-—.—.-
. ._ 5
1 . .u. IoNo
.....1
..nm
.m""— .V
J .... lovoe
Hm; mu.
..m mm
I ”u. 1.0de
.. N
1 3
1; 3
1 mm [00.0
- __
1 _ _ _ _ _ _

117

Examination of these Spectra reveals small changes in
absorbance in the wavelength region 300 nm - 450 nm and

a large growth, centered at 508 nm, correSponding to the
growth of the quinonoid complex. Although it is not
readily apparent from Figure 1, there is a Shoulder at
approximately 480 nm associated with this long wavelength
band (3). A Similar growth of quinonoid, but centered

at 502 nm was observed when alanine was mixed with trypto-
phanase (Figure 2). However, the growth at 502 nm was
accompanied by a slower growth centered at ~430 nm with
concomitant decay over the range 300 nm - 390 nm. A
clean isosbestic point occurred at ~390 nm as shown by
the difference Spectra in Figure 2. The absorption due
to quinonoid appeared to be unaffected by this Slower
process. The explanation for this phenomenon is unknown
and awaits further experimentation.

Analysis of the biphasic growth at 508 nm observed
with ethionine using equation 2 in KINFIT (17) demonstrated
that the first six to eight seconds of the growth could be
adequately described as the sum of two first order pro-
cesses, kl' and k2' (Figure 3). The rate constants k,‘
and k2' were the same at 470 nm, 480 nm, 485 nm, 490 nm,
500 nm and 510 nm, i.e. at several selected wavelengths
through the 500 nm absorption manifold. Data taken at

longer times revealed yet a third slower first order

118

Fi ure 2. Tryptophanase spectra collected as a
function of time after mixing with alanine at 24° C in
25 mM K-Epps, pH 8.0, 0.1 M KCl, 1 mM EDTA, 0.1 mM
pyridoxal-P and 0.2 mM DTT. After mixing: 0.225 M
alanine and 0.88 mg ml’1 tryptOphanase. The first
Spectrum has been substracted from all of the other
spectra to give rise to this set of difference spectra.

 

119

 

ll.-.00000" O. 00000.. O...

 

I1!
E‘UOIUO. 0.0.0
0-0000000000000000

 

I
I.‘.CO:O‘O.C

 

 

 

 

 

III‘II

 

llIIII
375 400 425 450
WAVELENGTH (NM)

lllll

l
350

 

0.50 *—

0.00T| ' I
O.IO

Q

 

550

500

330

FIGURE 2

120

F1 ure 3. The biphasic growth in absorbance at
508 nm after mixing tryptophanase with ethionine at
24° C in 25 mM K-Epps, pH 8.0, 0.2 M KCl, 1 mM EDTA,
10 0M pyridoxal-P and 0.2 mM DTT. After mixing:
10 mM ethionine and 1.0 mg ml"1 tryptophanase. The
solid line was calculated with equation 2 using the
parameters listed in Table III.

 

121

 

E [S

0.40

 

RBSURBRNC
0.20

 

 

 

I
1-00

2200
TIME (SEC)

FIGURE 3

3300

4100

122

growth. The majority of the results presented in this
paper deal only with the first two phases. The relatively
long times required to collect data during the third phase
made it technically difficult to define the fast phase in
those cases where the fixed wavelength mode was used.
Additional information available from the analysis
was the total absorbance change (here represented as the
sum of AA. and AA;) and the fraction of the absorbance
change attributable to each phase. Since data for this
experiment were collected for only 6 seconds the contri-
bution of the Slow third phase is not included. Assuming
that the percent of the contribution of the third phase
is independent of the concentration of ethionine, a
KD s 0.67 1 0.04 mM for the interaction of ethionine was
determined by a weighted least squares analysis as
suggested by Wilkinson (18), of the reciprocal of the
total AA at 508 nm given in Table l as a function of the
reciprocal ethionine concentration. This is in close
agreement with the value of 0.52 mM given by Watanabe
and Snell (8). Similar treatments of AA: and AA: gave
values for KD = 0.50 1 0.004 mM and 0.90 1 0.14 mM,
reSpectively. Of particular interest, however, was the
observation that under these conditions (pH, etc.) AA; and

AA: were roughly of equal amplitude.

123

Table 1. Kinetic parameters for the biphasic growth of
quinonoid as a function of ethionine concen-
tration. Values were obtained by fitting the
data for the change in absorbance at 508 nm
as a function of time to equation 2. Total
AA is defined as the sum of the absorbance
changes in the fast and Slow phases. Percent
AA: and AA; are the percent of the total

absorbance change attributable to each phase.

[L-ethionine] Total % %

(mM) k,‘(sec") k2'(Sec") AA AA, AA,
.30 2.3 i 0.023 0.38 i 0.004 0.172 70 30
.40 3.1 1 0.014 0.41 i 0.012 0.247 64 36
.75 4.4 1 0.017 0.42 1 0.008 0.340 59 41

1.0 5.2 1 0.081 0.42 t 0.001 0.381 56 44

1.5 7.2 1 0.064 0.44 1 0.004 0.452 54 46

5.0 14. 1 0.11 0.61 1 0.005 0.562 52 48

10. 17. 1 0.29 0.71 i 0.003 0.591 55 45
20. 18. 1 0.55 0.72 1 0.017 0.442* 57 43

* These data were collected at a slightly lower wavelength
and therefore the absorbance change was smaller.

124

In order to determine if k,‘ and k,’, the apparent first
order rate constants for the fast and slow phases, respec-
tively, showed any dependence on inhibitor concentration,
the biphasic rate of quinonoid formation at 508 nm was
examined over a range of ethionine concentrations from
0.3 mM to 20 mM. The results of these studies are presented
in Figure 4 and Table I. It can be seen from Figure 4 that
k;' exhibits a hyperbolic dependence on concentration while
k2' is essentially independent of ethionine concentration.
The fact that kz', that is the slow phase, is essentially
unchanged over this wide range of inhibitor concentrations
suggests that k,‘ reflects an enzyme conformational change.
When the data for k,’ from Figure 4 were plotted in recip-

l

rocal form, a value of 20.6 1 0.6 sec' was obtained for

kl' at infinite ethionine concentration.

Table II contains kinetic parameters for quinonoid
growth obtained with 50 mM, 100 mM and 200 mM alanine.
Although the dissociation constant for the interaction of
alanine with tryptOphanase (8) is not known precisely, it
appears that the three concentrations of alanine used were
saturating, or that k1' is insensitive to the concentration
of alanine, as no variation in k1' was observed. It is

interesting to note that although under saturating condi-

tions k,‘ for alanine is significantly slower than k,’

125

Figure 4. The variation of the apparent first order
rate constants, k,‘ (0) and k2' (A), for the fast and slow
phases, respectively, of the biphasic growth of quinonoid
with ethionine concentration. The conditions under which
these experiments were carried out are described in Materials
and Methods.

126

v mmauwu

122% .ozwuo mszoHrhm

 

 

 

 

0 o.N ._ . o. .m o. 0W4.
08

ll.—

0

no

1 #30
0.....—

HO

HO

...—U

1.1

1 133
03

0

N

S

:9 1M

_ . 4 . mUN

 

 

127

Table II. Kinetic parameters for biphasic quinonoid growth
at three concentrations of alanine. Values were
obtained by fitting the data for the change in
absorbance as a function of time to equation 2.
Total AA and percent AA: and AA; are defined in

Table I.
[L-alanine] . -1 . -1 Total % %
50 2.2 i 0.064 0.39 i 0.011 0.457 50 50

H-

100 2.2 i 0.12 0.44 0.028 0.317 50 50

H'-

200 2.7 i 0.12 0.59 0.044 0.351 49 51

* Differences in total absorbance change reflect the fact
that these data for the different alanine concentrations
were collected at slightly different wavelengths.

 

for ethionine, k2' is comparable for both inhibitors. This
is consistent with our previous suggestion that kz' reflects
an enzyme conformational change. Again with alanine the
fast and slow phases each account for roughly fifty percent
of the total absorbance change in the two phases.

Ehanges at 337 nm and 420 nm - Holotryptophanase exhib-
its absorption bands due to bound coenzyme centered at 337
nm and 420 nm whose relative amplitudes vary with pH (3).
Morino and Snell (3) associated the 337 nm form of trypto-
phanase with active enzyme on the basis that at pH values
above 8.0, where tryptophanase appeared to exhibit its
greatest activity, the 337 nm form predominated. In
addition, the more effective monovalent cation activators

appeared to stabilize the 337 nm form. The pH dependent

128

interconversion of the 420 nm and 337 nm forms had a pKa
of 8.14 1 0.06 in our hands which was at variance with
the value of 7.2 reported by Morino and Snell (3). Thus,
significant amounts of 420 nm absorbance exist at the
apparent pH optimum of 8.3 for tryptOphanase. Also, the
fact that we observed activity in the presence of LiCl,
which is essentially always in the 420 nm form prompted us
to investigate these enzyme forms in a more quantitative
manner in an attempt to determine the roles played by the
337 nm and 420 nm forms in catalysis. We reasoned that
by following the changes in absorbance at 337 nm, 420 nm
and 508 nm after mixing tryptophanase with ethionine in
the scanning stopped flow Spectrophotometer, we might be
able to correlate the rates of change at 337 nm or 420 nm
with the 508 nm absorbance and thus assess the importance
of each form in the activity of the enzyme.

Figures SA, B show the changes which occur in the
tryptOphanase spectrum at 420 nm upon addition of ethionine.
Recall that all wavelengths in the range 280 nm - 600 nm
are monitored in the same experiment. The change in
absorbance at 420 nm is composed of a fast first order
decay with an apparent first order rate constant of
18 t 2.2 sec"1 (see Figure SB) followed by a slow first
order growth with an apparent first order rate constant of

0.38 1 0.05 sec". Following the fast first order depletion

129

Bi ure 5. The changes in the tryptOphanase absorption
spectrum at 337 nm and 420 nm as a function of time after

mixing with ethionine in 25 mM K-Epps, pH 8.0, 0.2 M KCl,
1 mM EDTA, 10 uM pyridoxal-P, 0.2 mM DTT. After mixing:
10 mM ethionine and 1.0 mg ml'1 tryptophanase. A) The
overall absorbance changes at 420 nm; B) The rapid
decrease in absorbance at 420 nm; C) The overall absor-
bance changes at 337 nm. The solid lines were calculated
with equation 1 using the parameters listed in Table III.

 

-——«v-— _— 1.4

130

<m mxauuu

Humww m2:

 

 

85 8.4 SQ 8.00
F, P .

 

 

 

131

 

 

 

nu
nu

86 8.4 gum 850.
HZZQNVH OOH x muzqmmowmm

 

 

mu
my

 

 

FIGURE 5B

132

Um mxaomm

 

Humww m: H F
co m om Pv oo pm om. ha 00 5.1%
OS
00
HO
8
HO
ZN
L AU l. mlu
e m3
VA
0
1 I80
.0
0]
g
8
my Ila
N
i TVHW
— [

 

 

 

00'

133

of 420 nm absorbance, the 337 nm form of the coenzyme
disappeared slowly with an apparent first order rate
constant of 0.56 t 0.08 sec'1 (Figure 5C). In these
experiments values of k1' and k2' for the fast and slow
phases of quinnoind formation at 508 nm were 15. 1 1.2 sec"1
and 0.62 1 0.06 sec". respectively. Therefore, the rapid
phase of quinonoid growth (k1' = 15. 1 1.2 sec") occurs
simultaneously with a rapid loss of a 420 nm absorbing
coenzyme Species (kl' - 18. 1 2.2 sec"). This suggests
that the 420 nm form is the form poised for activity. The
observed slow increase at 420 nm following the fast phase
(k' - 0.38 t 0.05 sec“) presumably arises from what was
originally coenzyme in the 337 nm form being redistributed
among 337 nm, 420 nm and 508 nm absorptions according to the
new equilibrium which exists in the presence of ethionine.
Alternatively, this could be due to a build-up of_a 420 nm

absorber of unknown identity as was observed with alanine.

These data are summarized in Table III.

Effect of_pH on Quinonoid Formation - Morino and Snell
(3) demonstrated that more quinonoid is formed with alanine
as the pH is increased. A similar relationship was observed
with serine transhydroxymethylase (2) for the quinonoid
formed with D-alanine, and for tryptophanase with ethionine

(Figure 6). Because the amount of the 337 nm form and the

Table III.

134

Kinetic parameters for the absorbance changes
at 337 nm, 420 nm and 508 nm which occur when
tryptophanase is mixed with ethionine. The
data at 508 nm were fitted to equation 2. The
data at 337 nm were fitted to equation 1. The
data at 420 nm is composed of two parts as
described in the text. Each part was fitted
separately to equation 1. Total AA and percent
AA: and AAz are defined in Table I.

Absorbance changes at 508 nm:

 

 

k,'(sec") k2'(sec'1) Total AA Ail A22
15. t 1.2 0.63 1 0.058 0.747 58 42
Absorbance changes at 337 nm and 420 nm:
wavelength ' _

(nm) k (sec ‘) AA
337 nm 0.56 t 0.080 0.026 1 0.0012
420 nm (decay) 18. t 2.2 0.049 2 0.0010
420 nm (growth) 0.38 1 0.050 0.017 1 0.0004

135

Fi ure 6. The rate and extent of quinonoid formation
as a function of pH. TryptOphanase was pushed against
ethionine at three pH values as described in Materials and
Methods and quinonoid growth was monitored at 508 nm.
pH 7.2, (0); pH 8.0, (A); pH 8.7 (+). The solid lines were
calculated using the parameters listed in Table IV.

136

 

 

060 0230

-\ C
.v—.

 

4'50

2'00 2'50 3100 3'50 400
TIME (SEC)

I50

1

l
[00

I

0.50

C)

0

 

010 0390 03:0 030 03:0 050 0:0 00'
(WNBOQ) BONVBHOSBV

OF)

FIGURE 0

137

420 nm form depended on pH and since the 420 nm form appeared
to be poised for activity, we examined the rate of quinonoid
formation and the amount of quinonoid formed at equilibrium
when tryptophanase was mixed with 5 mM ethionine at pH 7.2,
8.0 and 8.7 as described in Materials and Methods. The data
at each pH which were collected for a period of 16.5 seconds,
were analyzed as the sum of three exponentials, the first
two phases of which are presented in Figure 6. The kinetic
constants and amplitudes for all three phases are summarized
in Table IV.

Examination of Table IV reveals that as the pH is
increased, kl' also increases, from 8.7 1 0.28 sec'1 at
pH 7.2 to 13. t 0.89 sec"1 at pH 8.0 and 19. 1 2.3 sec'1
at pH 8.7. In contrast, kz' and k,‘ appear to be relatively
insensitive to pH. This is consistent with the notion that
kz' and k,‘ reflect enzyme conformational changes.

The relative amplitudes attributable to each phase
given in Table IV are also of interest. As the pH is
increased the contribution of the fast phase to the total
absorbance change diminishes, while the second and third
phases contribute more.

TryptOphanase-ethionine (minus cations) VA NH.C1, KCl,

RbCl and CsCl - Suelter and Snell (9) have shown that

 

ethionine interacts with tryptOphanase in the absence of

cations, presumably forming a Schiff's base with enzyme

138

 

o~ om vm oom.o om.o n om.o om.o « H.H m.N a .ma 5.»
ma ow Ne moo.c o~c.o w woo.o cec.c H ~5.c ow.c a .ma o.w
o“ NN we Hoe.o euo.o n «H.o owo.o u eu.o m~.o n u.» ~.>
.<< ~<< .<< << A.-uomv..x A.-uomv.~x A.-uomv..x ma
w w » Hooch
.omoaa :uoo ou oHnousnwhuuo omconu
oucoAHOmao Hope» on» we «coupon on» own n<< paw «<4 ..<< unouuom
.momonn oohnu on» a“ momcocu ooconuomno oau mo Sam ogu mo cocwmov
ma << Houoe .maowucocomxo oougu we saw onu wcfi>ao>cw N on uofiwswm
newuosuo no on oswu mo :owuocsm o mo 5: «cm no ouconuomao cw omconu
oau how meow ozu wcwuuwm x: vocwouno ouoz mo=Ho> .h.m paw o.m .~.s
mosao> :9 an vfiococwsc mo suzoum owmonmwuu onu you muouosouoa uwuocwx ”>H oHnoh

139

in the a-conformation. However, no quinonoid is formed
until activating cations are added. In an attempt to gain
more information on the role of cations in this process,
tryptOphanase was prepared as described in Materials and
Methods in the presence of 10 mM ethionine and (CH,).N-Epps
in the absence of activating cations.

Figure 7 shows the results obtained when this enzyme
solution was pushed against saturating concentrations of
NH,+, K+, Rb+ and Cs+. Quinonoid growth at 508 nm was
again biphasic. The kinetic parameters obtained for each
cation are given in Table V along with the percentages of
the total absorbance change attributable to the fast and
slow phases.

The amount of quinonoid formed at equilibrium was

+ and Rb+, but consider-

approximately the same in NH.+, K
ably less in C51 which is consistent with what was pre-
viously shown (9). However, the percentage of the total
absorbance change occurring in the fast phase is larger
in the case of the more effective monovalent cation
activators. Values of k1' and kz', the apparent first
order rate constants for the fast and slow phases,

respectively, appear to increase as the effectiveness

of the activator decreases.

140

Fi ure 7. The rate and extent of quinonoid formation
in the presence of various monovalent cations. A trypto-
phanase solution containing ethionine was prepared in the
absence of activating cations as described in Materials
and Methods. This enzyme solution was pushed against
saturating concentrations of four monovalent cations and
quinonoid growth was monitored at 508 nm. 0.025 M NH.C1,
(0); 0.1 M KCl, (A); 0.25 M RbCl, (+); 0.5 M CsCl 00).

The solid lines were calculated with equation 2 using
the parameters listed in Table V.

 

141

 

 

 

 

:50 2'00 2.50
TIME (SEC)

1'00

0'50

 

 

020 0'20 o'io
WN 809 (BONVBHOSBV) V

7

FIGURE

142

 

mu BN wwH.o mNo.o a wm.o mm.o w m.m om.o +mu
we on oo~.o nmo.c H we.o sm.o w m.m m~.c +am
Hm av un~.o NHo.o n ev.o Hmo.o u v.~ oH.o +2
cm on moN.o mao.o n Hm.o omo.o n w.~ m~o.c +.:z
c:
~¢< ~<< <4 a~-uomv.~x Aduuomv.~x :owuouu coauou
» « Hooch -coonou
.H oanoh a“ vocwmop ouo ~<< woo .<<
acouuon can << annoy .N :owuosco on oafiu mo :owuocsm m on a: mom um
ouconuomno :M omconu onu how sump o:u mcwuuwm >9 vocflouno ouoz mosao>
.mcofiuoo ucoao>ocos new; uost ma xoaasoo ocficownuo-omoconnoaaxuu o
cog: vo>uomno pflococwzc mo nuzouw oflmmgnwn on» you muouoeonon aduocmx .> oHnoe

143

Deuterium Isot0pe Effect on Quinonoid Formation -
Figure 8 shows the results obtained when tryptOphanase was
mixed with L-[a-‘H]alanine and L-[o-ZHJalanine. Substitu-
tion of deuterium at the a position of the inhibitor effects
both the extent and rate of quinonoid formation. At equi-
librium, approximately 4.5 times more quinonoid is formed
from L-[o-’H]alanine than from L-[a-2H1alanine. The
progress curve for quinonoid formation from L-[o-‘H1alanine
was biphasic giving a value for k1' of 2.2 t 0.02 sec'1
and 0.38 1 0.003 sec'1 for kz'. On the other hand, with
deuterium at the a position, the two apparent first order
rate constants, k1' and k2', were too close in value to
be separated by our curve fitting procedures. Therefore,
these data were fitted to a single exponential giving a
value for k' or 0.49 t 0.005 sec". It thus appears that
the effect of deuterium substitution is to slow down the
fast phase of quinonoid growth while leaving the slow
phase virtually unchanged. This is interpreted as additional
evidence that k,’ reflects an enzyme conformational change.
The deuterium kinetic isot0pe effect of approximately four-
fold also shows that loss of the o-proton is the rate-
limiting step when quinonoid is formed from the competitive

inhibitor of tryptOphanase, L-alanine.

144

Fi ure 8. The effect of deuterium substitution at the
o-position of alanine on the rate and extent of quinonoid
formation with tryptOphanase. Try tophanase was pushed
against 0.45 M L-[o-‘H1-alanine (0 and L-[o-zfll-alanine
(A) as described in Materials and Methods. The t0p line
was calculated with equation 2 using the values: A, - 0.70,
AA; = 0.43, AA, = 0.22, k1' = 2.4 sec", and kz' - 0.38 sec".
The bottom line was calculated with equation 1 using the
values Aco = 0.17, AA = 0.14, and k1' - 0.49 sec".

 

145

00 .m 00¢

x 5:5 _ m

00.0

 

 

 

 

Cmmv NEE.
00m com 00..
_ _ _

I

o
(x!
o

. . coo

. 0.0

J

o
'9.
o

l
o
g
(INN 209) BONVBHOSBV V

l
o
'n.
o

I. 00.0

 

05.0

 

146

DISCUSSION

The results presented in this paper are consistent with
a model of tryptOphanase catalysis based on previous equili-
brium and stopped flow studies on the effects of pH and
monovalent cations on the spectral and catalytic pr0perties
of the enzyme. Scheme I is a representation of this model
which involves four enzyme conformations, a, B, y, and 6
defined previously.

Bthionine and alanine were shown to interact with
tryptophanase to form quinonoid derivatives absorbing at
approximately 500 nm (Figures 1 and 2). The appearance
of quinonoid as a function of time for the first six to
eight seconds of the reaction was composed of a fast and
a slow first order process with rates represented by k,‘
and k2', respectively. The relative amplitudes of each
phase at pH 8 were approximately equal with both ethionine
and alanine (Tables I and II), although in the case of
ethionine it appears as if a slightly greater percentage
of the absorbance change in the two phases of quinonoid
growth occurs in the fast phase. Examination of data
presented earlier (Section 2, Table III) reveals that at
pH 8.0 approximately 56 percent of the enzyme, in the
absence of substrate or inhibitor, is in the B conformation

while approximately 45 percent is in the y conformation.

147

 

H mes—Um

 

 

 

 

 

 

...
+1 z 2
O O _ _
gov .. 101.. 4%.... _
L -«00xwuzu-.. 3+ ..7
. +

 

 

 

 

 

 

148

The results presented in this paper for the biphasic forma-
tion of quinonoid at pH 8.0 are consistent with Scheme I
where inhibitor interacts with the B conformation to rapidly
form the quinonoid derivative with an apparent rate, k,’.
According to this interpretation, the slower phase, kz',
represents the conversion of conformation y to the B
conformation. Enzyme in the B conformation, which is
assumed to represent the active form of the enzyme, could
thus rapidly form additional quinonoid with an apparent
rate, kz', dictated by the rate of the conformational change.

This suggestion is supported by the fact that k2' is
essentially unaffected by inhibitor concentration (Figure
4) or the nature of the inhibitor which is consistent with
a change in enzyme conformation. In addition, the average
values of k,' of 0.51 1 0.14 sec" and 0.47 2 0.15 obtained
with ethionine (Table I) and alanine (Table II), reSpectively
agree closely with the value for the rate of conversion of
conformation y to conformation 8 following a rapid decrease
from pH 8.70 to 8.25 of 0.52 t 0.02 sec.1 (Section II,

Table I).

The changes in absorbance at 337 nm and 420 nm which
occur when ethionine is mixed with tryptOphanase are also
consistent with Scheme 1. The rapid disappearance of 420 nm
absorbance coincides with the fast phase of quinonoid forma-

tion and suggests that the B conformation, containing Species

149

of coenzyme which absorb at approximately 420 nm, is the
active form of the enzyme. The disappearance of 337 nm
absorbance occurs with essentially the same rate as the
second phase of quinonoid growth. This implies that the
y conformation or 337 nm form of tryptophanase cannot form
quinonoid directly but must first be converted to the B
conformation before the a-proton can be removed from the
inhibitor.

Although interpretation of the data for the growth
of quinonoid at pH 7.2, 8.0 and 8.7 is complicated by the
fact that the dissociation constant for ethionine increases
as the pH is decreased, the variations in the rates and
amplitudes of the three phases of quinonoid growth are
again consistent with Scheme I. At lower pH values where
the enzyme exists primarily in the B conformation (Section
2, Table III) relatively more quinonoid is produced in the
fast phase (Table IV), which supports the suggestion that
this is the active form of the enzyme. The second and third
phases correSpond to the slow conversion of conformation y
and 6, respectively, to conformation B, with subsequent
rapid quinonoid formation accompanying the establishment
of a new equilibrium between conformations in the presence
of ethionine. The observation that the second and third
phases of quinonoid growth account for a greater percentage

of the total absorbance change at 508 nm as the pH is

150

increased follows from the fact that more of the enzyme
is in the y and 6 conformations at higher pH values. Within
experimental error, only the rate of the first phase, that

is the formation of quinonoid, 1308 from HIE is affected

8’
by pH, increasing from 8.7 t 0.28 sec" at pH 7.2 to 19. 1
2.3 sec'1 at pH 8.7. The rates of the second and third
phases appear to be constant with pH which again is consis-
tent with the proposed conformational changes in Scheme 1.
In the absence of activating cations, tryptophanase
is inactive and absorbs at 420 nm (3). We have assumed that
monovalent cations act at or near the catalytic site in
close proximity to the coenzyme (9) and that in the absence
of cations the coenzyme is held in a slightly different
conformation in relation to enzyme functional groups
required for catalytic activity. We have designated this
inactive, 420 nm absorbing form of the coenzyme conforma-
tion a. Scheme I shows conformation a (Ca) capable of
being converted to the conformations B, y, and 5 by mono-
valent cations. In the absence of cations it is assumed
that ethionine forms a Schiff's base with conformation o (9)
(not shown in Scheme I), but nothing else is known about
this complex. In an attempt to obtain information on this
interaction and possibly determine the rate of conversion
of Co to CB we pushed an enzyme-ethionine complex without

activating cations against saturating concentrations of

151

NH.C1, KCl, RbCl and CSCl as described in Materials and
Methods. The results presented in Table V and Figure 7
show that the extent of quinonoid formation was approximate-
ly the same in NH.’, K+ and Rb‘ but less in CS+. These
results are consistent with those of Suelter and Snell (9)
who found that the amount of quinonoid formed at equili-
brium at pH 8.0, was prOportional to the effectiveness of
the cation as an activator. As mentioned, the values of
k,’ and kz', the apparent first order rate constants for
the fast and slow phases of quinonoid growth with the
various cations, appear to increase as the effectiveness
of the activator decreases. Since this increase in rates
also parallels an increase in ionic strength from 0.025 M
in the case of NH,+ to 0.5 M in Cs’, a full explanation

of the effect of cations on quinonoid formation must await
further studies on the effects of ionic strength on this
process. Likewise, although the value of k,‘ may reflect
the rate of conversion of Ca to CB prior to rapid quinonoid
formation form the CB conformation, the unavoidably low
specific activity of the enzyme which resulted from
dialysis in the absence of activating cations makes firm

conclusions difficult.

152

Finally, Figure 8 shows that the substitution of deu-
terium at the a-position of alanine causes a dramatic
decrease in both the extent of quinonoid formation and
the rate of the fast phase of the biphasic quinonoid
growth. These results are consistent with Scheme I if
it is assumed that the fast phase of quinonoid (EQB)
formation occurs through ESB from HIEB. The observed
kinetic isot0pe effect on kr', the apparent first order
rate constant for the fast phase implies that proton

abstraction is the rate-limiting step in quinonoid for-

mation with alanine.

10.

11.

12.

13.

14.

15.

16.

153

REFERENCES

Jenkins, W. T. (1961) J. Biol. Chem. 236, 1121-1125.

 

Schirch, L., and Jenkins, W. T. (1964) J. Biol. Chem.
239, 3801-3807.

Morino, Y., and Snell, E. E. (1967) J. Biol. Chem.
242, 2800-2809.

 

Fonda, M. L., and Johnson, R. J. (1970) J. Biol. Chem.
245, 2709-2716.

 

Ulevitch, R. J., and Kallen, R. G. (1977) Biochem.
16, 5350-5354.

Kumagai, H., and Yanada, H. (1970) J. Biol. Chem.
245, 1773-1777.

 

Walsh, C. (1979) Enzymatic Reaction Mechanisms,
W. H. Freeman and Co., San Francisco, Chapter 24.

 

Watanabe, T. and Snell, E. E. (1977) J. Biochem. 82,
733-745. -—'

Suelter, C. H. and Snell, E. E. (1977) J. Biol. Chem.
252, 1852-1857.

 

Suelter, C. H., Wang, J., and Snell, B. E. (1977)
Anal. Biochem. 16, 221-232.

 

Suelter, C. H., Wang, J., and Snell, E. E. (1976)
FEBS Lett. 66, 230-232.

 

Hngerg-Raibaud, A., Raibaud, 0., and Goldberg, M. E.
(1975) J. Biol. Chem. 250, 3352-3358.

 

Coolen, R. B., Papadakis, N., Avery, J., Enke, C. C.,
and Dye, J. L. (1975) Anal. Chem. 41, 1649-1655.

 

Papadakis, N., Coolen, R. B., and Dye, J. L. (1975)
Anal. Chemo fl, 1644-16490

 

Suelter, C. H., Coolen, R. B., and Dye, J. L. (1975)

 

June, D. S., Kennedy, B., Pierce, T. H., Elias, S. V.,
Halaka, F., Behbahani, Nejad, I., El-Bayoumi, A.,
Suelter, C. H., and Dye, J. L. (1979) J. Amer. Chem.
Soc. 121, 2218-2219.

154

References - continued

17. Dye, J. L., and Nicely, V. A. (1971) J. Chem. Ed.

 

48, 443-448.
18. Wilkinson, G. N. (1961) Biochem. J. 80, 324-332.

 

APPENDICES

155
APPENDIX A

Derivation of the eXpression for the rate of intercon-
version of the B and Y conformational manifolds follow-
ing a rapid change in pH (refer to Section II, Scheme I

for the mechanism).

The differential equation describing the disappearance
of the Y conformational manifold following a rapid

decrease in pH is

-d H‘EY + E
(I a: ' 1') - k,,[EY]-k1[EB]+k,[H+EY]-k-3[H+EB]

from equilibrium considerations,

- . + 3 H+

[be] mums] (2) and [By] W. (3)
[H’] [H ]

therefore,

 

'd([H+EI] * (Ell) . _ 1 +_£1 d[H+Eyl
dt [HI] T

= k 1K»
—=—;— . k [HIE 1 - (k *kao)
( [H ] a) Y [H 1 [H E 8]

(4)

156

from conservation conditions,

 

 

[H‘BBI + [“5] + [H‘EY] + [By] - [E.,] (5)
or,
[111.15 ] (1 1 K0 )4- [H‘E] 1 + K.,

8 [11+] Y BIT-i. - E0 (6)
solving for [HIEB] in terms of [Ba] and [HIEY] we obtain
[H+EB] . [2.1 - IH+EYJ (1. K. W

(1 1 K0 ) [11+] 1!
[HI] (1- K, ) I (7)
[3*] )

substitution of this expression for [HIEB] into (4) gives

1 + K, d[H*By] k ,x
‘ —-—- ‘ " +k HIE + k + lel
( I'm) '1' "H” ')[ Y] (-3 [11+] ) x

‘f(k-3 * k1K0)1
D [H‘] 4}
[£0] (3)

1 + K0 ' IH‘EYJ - 1 1 + K0
1' E—I)‘ L1 [11*] )3

 

 

 

 

 

 

 

 

157

 

 

 

 

 

 

 

 

 

simplifying,
-d[H+Bxl . k'a+ k1K° E k-1K4 + k k_3+k1Ko
dt IH*1 [ “1 [n+1 ’ [9+] IH‘EYI
+ +________
1+x, 1+1<u 1+x, )Q+K°)
a b
(9)
Equation 9 has the form,
d[H+EY] .
._ = a - b[H E ] (10)
dt Y
let

y = a - b[H*EY] - d[H*EY]

dt

then,

dy

.__ . - d[H*E 1

dt b (‘ Y ')

dt

therefore,

dy

dt ' 'bdt

integration gives

Y ‘ hie-bt

B.

158

Therefore the apparent first order rate constant for
the conversion of the y to the B manifold is equal to
the expression for b given in equation 9. The
expression is the same for the conversion of the y to
the 3 manifold. The parameter k-, may be eliminated
from the expression for b through the use of the

thermodynamic 100p constraint
k-a ' Ko k1
K“ it, k3

Derivation of the expression for the rate of appearance
or disappearance of 56 following a rapid change in pH
(refer to Section II, Scheme I for mechanism). The
differential equation for the disappearance of E6

following a rapid decrease in pH is

d E
A , 1-2[55] - k, [55]

dt (1)
The equilibrium conditions are
x. - [Eg][H*1 , x. - [Ex] . K2 . [55]
[HIEBI [EB] ’ TIE—T

159

K. = [H'Eg] K. - [EyllHI]

#

[H+By]

 

+

H E

[ Y1
The enzyme conservation equation is

[E0] = [H*531 + [EB] + [H‘Byl + [Ev] + [2,]

= . n+ +
[By] (1 + _1_ + [ l + [H] +035] (2)
K1 KOKI KOKIKS

 

 

therefore,
[E7] ' [B0] ' [By]

(1 + .1. [HI] [HI] 3 (3)
+ +.______.
K! Kok. K.K.K3

 

 

Let (I + _1_+ [11+] + [11+] ) equal D, then
K1 KOKI KoK1K3

[By] = [so] - [56] (4)

D D

Substituting this expression for [56] into equation 1 gives

d[E ] k [E 1
—d%" 2n o .(1132 Ik'2)[55] (5)

Let

y g kZIEo] k2
D ’ 3‘ + k.2 [E6]

 

 

 

160

 

then,
dy k d[E5]
dt (D * k...) dt (61
or
32" = - £3 + k )d (7)
y D '2 t

integration of equation 7 gives

'(‘l'g‘z‘l‘ k-2)t
Y = Yoe
thus,
k2
'5'+ 1.-.

is the expression for the apparent first order rate constant
for the disappearance of E5 following a sudden decrease in
pH. This is also the expression for the appearance of E6

following a rapid increase in pH.

161

APPENDIX B

DOCUMENTATION OF PROGRAM MPLOT.

MPLOT was deve10ped in response to the need for the
inexpensive construction of graphs of publishable quality.

The program enables the user to plot from one to five
curves comprised of pairs of X, Y coordinates on a single
set of axes using the MSU CalComp Plotter. In addition
to the plotting of conventional X-Y data, MPLOT has the
capability of calculating and plotting theoretical curves
through selected data curves.

Usage:

A. Construction of conventional X-Y plots

The program MPLOT is maintained as a permanent file on
the MSU CDC 6500 computer. It can be accessed via the
subroutine THEQN as described below. For this application
THEQN is a dummy subroutine and its contents will be ignored
by MPLOT. The appropriate data cards are simply appended
to the THEQN deck and the job is ready to be submitted.

B. Drawing theoretical curves through data

The major difference between this and the previous
application is that a theoretical equation, describing the
theoretical curve to be drawn, must be included in the
subroutine THEQN. MPLOT will then use this equation along
with the values of XMINTH(I) and XMAXTH(I) to calculate
and ultimately construct the theoretical curve(s).

Deck Structure for the Use of MPLOT:

PNC
JOB CARD
PW I
ATTACH (MPLOT,MPLOT)
FTN.
LOAD, LGO.
?§LOT.
THEQN DECK
(73

9)
DATA CARDS

(6789)

Data Cards:

 

1.

ITITLE (8A10)
ITITLE

LABELX (A26)

LABELX

LABELY (A26)
LABELY

162

A comment card. This comment will
appear on the printout, not on the
graph.

The label which will appear on the
x-axis. Note: Center the label
in the 26 character field.

The label which will appear on the
y-axis. Note: Center the label
in the 26 character field.

NCURVS, IQUEUE, LINTYP, XAXLENG, YAXLENG, FACT, IBORDER,
ISET (315, 3510.4, 215)

NCURVS

IQUEUE

LINTYP

XAXLENG

YAXLBNG

FACT

IBORDER

The number of data curves on the
graph.

The QUEUE to which the plot is
submitted. Possible values of
IQUEUE are 0,1,2,S,6, and 10. [see
CDC 6500 User's Manual, Vol. VII].

If LINTYP - l, the data points for
each data curve will be connected
by straight line segments. If
LINTYP f 1, no straight lines will
be drawn (Default - -l).

The length of the x-axis in inches
(Default - 3.0 inches).

The length of the y-axis in inches
(Default - 3.0 inches).

Alters the size of the entire graph.
For example, a value of 2.0 for FACT

would produce a graph 6 inches
square using the default values of
XAXLENG and YAXLENG (Default - 1.0)
[see CDC User's Manual, Vol. VII
under "Factor"].

If IBORDER - l, a border will be
drawn around the graph. If IBORDER
f I, no border will be drawn.

¢S.

*8.

163

ISET If ISET = 1, user will set axes
dimensions.

XMINFV, XDELTAV, YMINFV, YDELTAV (4510.4)

XMINFV Smallest value to appear on x-axis.

XDELTAV Increment between tick marks on
X'aXis o

YMINFV Smallest value to appear on y-axis.

YDELTAV Increment between tick marks on
y-axis.

(ITHEQ(I), I - 1, NCURVS) (515)

ITHEQ(I) If ITHEQ(I) - 1, a theoretical
curve will be plotted through data
curve I. If ITHEQ(I) f 1, no theo-
retical curve will be drawn.

(NPTS(I), I = 1,NCURVS) (515)

NPTS(I) The number of datum points compris-
ing data curve I.

XMINTH(I), XMAXTH(I) (2510.4)

XMINTH(I) The smallest value of x for theo-
retical curve I.

XMAXTH(I) The largest value of x for theo-
retical curve I.

For each data curve J: (X(I,J), Y(I,J), I a 1,NPTS(J))
(8E10.4)

X(I,J) x coordinate of datum point I on
data curve J.

Y(I,J) y coordinate of datum point I on
data durve J.

Notes: 1. Data for each curve J begins on new card.
2. A maximum of 100 data points is allowed
for each data curve.

 

¢ Include this card only if ISET - l.
* Include cards only for each data curve through which a

theoretical curve will be drawn. Do not include any cards
if no theoretical curves are to be drawn. Zero to five
cards could appear here.

HICHIGRN STRTE UN V. I

I 1'11!“ H) mm I) will)

31006326

Rnazss
IIIIIIIHI
1153