THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 237, No.

7,July 1962
Printed in U.S.A.

A Kinetic and Equilibrium Analysis of the Glutamic Oxaloacetate Transaminase Mechanism*

SIDNEY F. VELICK AND JOHN VAVRA

From the Department

of Biological

Chemistry, (Received

Washington for publication,

University January
Aspartate

School of Medicine, 22, 1962)

+ ketoglutarate F;

St. Louis

IO, Missouri

The amino group carrier function of the coenzyme of transamination was postulated on the basis of coenzyme properties and model experiments (1, 2) and was established for the enzymatic reaction by the observation that pyridoxal and pyridoxamine phosphates exhibit equivalent activity in the reactivation of crude apoglutamate oxaloacetate transaminase (3) and by direct spectrophotometric observation of the coenzyme interconversion in experiments on the purified enzyme (4). The enzyme is stable and highly active and catalyzes a reaction that is susceptible to detailed kinetic analysis by continuous optical methods. Moreover, the bound coenzyme exhibits spectral changes that make it an indicator of events occurring in experiments carried out with substrate level concentrations of enzyme. It is therefore possible to attempt the correlation of mechanism and intermediary reaction equilibria, deduced kinetically, with results obtained by examination of the bound coenzyme itself under various conditions. The glutamate oxaloacetate enzyme is a mechanistic prototype for a large number of experimentally less accessible transaminases and also for the general class of group-transferring enzymes, which operate exclusively through binary enzyme substrate complexes. Experimental Procedure

glutamate

+ oxaloacetate

(1)

Enzyme and Substrates-Pig heart enzyme preparations were made by the method of Jenkins, Yphantis, and Sizer (4) and by modifications thereof, which yielded additional purification and produced an enzyme that approached homogeneity. Purification steps beyond the ammonium sulfate fractionation of the heated extract did not affect the kinetic behavior of the enzyme, nor was purification beyond the level obtained by these workers essential for the spectrophotometric analysis of the bound coenzyme. A physical and chemical description of the enzyme will therefore be reserved for a separate communication. The substrates were of reagent grade and were checked chromatographically for amino and keto acid contaminants. Except for oxaloacetate, solutions of which were prepared shortly before use, concentrated stock solutions of the substrates could be neutralized and stored in the frozen state. Stock enzyme solutions, dialized against 0.025 M arsenate buffer, pH 7.4, were stable for long periods of time. The amount of enzyme used in a kinetic test was of the order of 0.04 pg per ml. Initial Velocity Measurements-The transaminase catalyzes the equilibrium of Equation 1,
* This work was supported in part and H-22 of the United States Public by Research Grants Health Service. H-2732

which may be studied kinetically in either reaction direction by measuring the increase or decrease of absorbancy in the 260 rnp absorption band of oxaloacetate. In the neutral pH region and in the absence of metal ion contaminants, the absorption coefficient of oxaloacetate is 0.57 rnM-l cm-l, at 280 m,u, based upon weighed samples of the substrate and also upon enzymatic assay with an excess of reduced diphosphopyridine nucleotide and malic dehydrogenase at pH 6.8. Since the absorption coefficient is relatively small, it was necessary to use reaction cuvettes of lo-cm light path and a strip chart recorder with compensating circuits and a 5-fold expanded scale in order to obtain reliable initial velocities at low substrate concentration. By these devices, an absorbancy change of 0.02 gives a full scale (11-inch) recorder deflection. This sensitivity was more than adequate to cover the required range of amino acid substrate concentrations and just barely sufficed for the keto acids, which have Michaelis constants in the range from 0.03 to 0.1 mM. Reaction mixtures were made up to 25 ml, and reaction was initiated by addition of 0.05 ml or less of a standardized enzyme solution. Initial velocities were based upon the early linear portions of the reaction curve, usually involving the first few per cent of the total reaction. Brief initial lags were observed. These were most pronounced at very low substrate concentrations and at extreme pH values. They could not be accounted for consistently by the occurrence of rate-limiting enolizations of oxaloacetate, since they occurred in both reaction directions. Essentially the same Michaelis constants for aspartate and ketoglutarate were obtained by the assay system described above and also by measurement of oxaloacetate production at 340 rnp with an excess of malic dehydrogenase and reduced pyridine nucleotide, although the apparent maximal velocities in the latter case were somewhat larger. Kinetic Theory

Formulation of Reaction-The general formulation of the reaction is based upon the following observations. (a) The bound coenzyme undergoes cyclic interconversions between the amino and aldehyde forms, and (b) the enzyme catalyzes amino group exchange between glutamate-CL4 and unlabeled ketoglutarate in the total absence of the aspartate oxaloacetate pair (5, 6). These results indicate that the reversible amino group transfer between aspartate and ketoglutarate is the sum of two halfreactions (Equation 2).

2109

2110 Aspartate + aldehyde enzyme $

Glutamic Oxaloacetate Transaminase Mechanism

@a)

Vol. 237, No. 7

reactive enzyme species are in a steady state during initial velocity measurements. Derivations have also been made for the cases in which fewer intermediates are explicitly postulated Amino enzyme + ketoglutarate $ in the half-reactions. It turns out that the form of the rate @b) equations is independent of the number of postulated interglutamate + aldehyde enzyme mediates. For this reason, and because the algebra will be The sequences of intermediates begin with the formation of a greatly simplified, the initial treatment of the experimental complex between substrate and enzyme protein and proceed results will be based on what we term the minimal mechanism through the intramolecular formation of a Schiff base between (Equation 3)) in which only one intermediate is explicitly postusubstrate and bound coenzyme, a shift of the double bond to the lated in each half-reaction. The symbols, A, QI, G, and Ox refer opposite side of the imino nitrogen in the Schiff base, cleavage to concentrations of aspartate, ketoglutarate, glutamate, and of the isomerized Schiff base, and dissociation of amino or keto oxaloacetate, respectively. E and E are the functionally nonacid product from the protein. The simplest hypothesis condissociable complexes of pyridoxal and pyridoxamine phosphate cerning the intermediary complexes is that the substrates occupy with enzyme, and EX and EY are symbols for sequences of the same catalytic site one at a time in sequence, forming binary intermediates. enzyme-substrate complexes exclusively. This is the mechakl Ja Ox + E nism that is supported by the experimental results. A+.?#----+ EX (3a) kz h Derivation of Rate Equations-Since both half-reactions are freely reversible, the general formulation of the kinetic mechaks h ol+EEY-G+E nism with four intermediates in each half-reaction involves the (3b) kG ks explicit use of 20 individual rate constants. Derivations for this case have been made by the schematic matrix method of King The forms taken by the rate equations for the forward and and Altman (7) for the condition that involves the minimal reverse reactions are the same, since the equilibrium is symassumptions concerning the rate constants, namely, that the metrical, and are given in reciprocal form in Equation 4 oxaloacetate + amino enzyme
TABLE

EO -=Vf

;
f (

%+?+I
01 >

(44

Michaelis

constants of minimal
KA

and maximal velocities

mechanism
Kl

in terms of rate constants oj transaminase reaction

EO -=vr

r (

z+K&+l >

Mb)

Vfh klh

kd

Vr(ks ksk,

k7)

Vr(ks + k7) k&s

Vr(kz + kd k&4

kzks v, = ___ kz+ kg

where v and V are initial and maximal velocities, the subscripts, f and,, refer to forward and reverse reaction directions, and the Ks are Michaelis constants. As stated previously, Equation 4 is obtained both for the minimal mechanism and for the expanded mechanism containing any number of intermediates. The differences arise only in the expressions obtained for the Ks and Vs in terms of the individual rate constants. These expressions for the minimal mechanism are summarized in Table I. It so happens that the apparent association or dissociation constants of the enzyme-substrate complexes, corresponding to rate constant ratios of the type, k&, k&, etc., can be evaluated experimentally. At a later point in the discussion, we shall consider the meaning of these ratios in terms of the expanded kinetic mechanisms. It should be noted here that since the reaction is a cyclic one it makes no difference which of the two half-reactions is written first. Changing the order puts different numerical subscripts on the ks, but the proper identification of the Its is automatic in the analysis of the experimental data.

Kinetic Parameters
Forward and Reverse Reactions-According to Equation 4, a double reciprocal plot of initial velocity against the concentration of one substrate, 81, at a series of fixed concentrations of the second substrate, &, should yield a set of parallel lines, one for each concentration of 82. This is observed for both reaction directions (Figs. 1 and 2). Similar plots from the same sets of data are obtained with either substrate as the independent variable. The intercepts on the ordinates are reciprocals of apparent maximal velocities, l/V, and the intercepts on the abscissas are negative reciprocals of apparent Michaelis con-

FIG. 1. Double reciprocal plot of initial velocities against ketoglutarate concentration at a series of fixed concentrations of aspartate at pH 7.3 in 0.04 M sodium arsenate at 26.

July

1962

X. F. Velick and J. Vavra

2111

stants from Table I. Substitution of the numerical values of the maximal velocities and rate constants from Table II gives K = 6.2, which is in good agreement with the directly determined value and provides an independent test for the type of kinetic mechanism and the self-consist.ency of the kinetic parameters. The occurrence of Vf and Vr to the second power makes the Haldane relation a fairly sensitive criterion for this mechanism. Half-reactions-A distinctive property of the transaminase reaction is the sequential occurrence of two mutually inverted half-reactions. The occurrence of the half-reactions in the complete reacting system is established by the over-all kinetics,
I I I I I I I I I

40 80 /OXALACETATEhnM)

120

160

FIG. 2. Double reciprocal plot of initial velocity against oxaloacetate concentration at a series of fixed concentrations of glutamate, pH 7.3, in 0.04 M sodium arsenate at 26. slants, 1 /K,, . These quantities are related to the concentration independent constants by Equation 5. and G,
1

= -

-10

-5

5 IO 15 20 kKETOGLUTARATEhnM)

25

Accordingly, a plot of 1 /V against l/(Sz) yields V and Ksz, and a plot of l/Ks, against l/(82) yields K,, (Figs. 3 and 4). Data obtained in this manner for all four substrates are summarized in Table II. It is noted that the enzyme reacts more rapidly with glutamate and oxaloacetate than with aspartate plus ketoglutarate and that the Michaelis constants for the keto acids are much smaller than those for the amino acids. The concordance of the graphical form of the results with the requirements of Equation 4 established the validity of the kinetic mechanism of Equation 3 or of the equivalent expanded mechanisms. If ternary complexes between enzyme and two substrates were formed, then the rate equations would contain terms of the type, Ks,Ks,/(X1)/(S2), and the plots corresponding to Figs. 1 and 2 would be not sets of parallel lines but sets of lines that intersected in a left-hand quadrant. This is what occurs with pyridine nucleotide-linked dehydrogenases, which form ternary complexes in which the coenzyme behaves as a second dissociable substrate. The reactive complex in transamination is actually ternary if we include the coenzyme, but the coenzyme in this case is not dissociable and hence is not an independent variable. The above interpretation of the kinetic mechanism is also supported by the experiments described in the following sections, which deal with substrate and product inhibition, equilibria, and various kinds of competitive inhibitors.

FIG. 3. Secondary plots from data of Fig. 1. I, Graphical analysis yielding l/K, as intercept on the ordinate. KA is the quotient, slope/intercept (Equation 5). II, Graphical analysis
as intercept on the ordinate at two different enzyme and -1/K, as intercept on the abscissa. The same data, replotted as in Fig. 1 with aspartate as the independent variable and anal.yzed as in Fig. 311, .yield -l/K* and the same l/VI. yielding l/Vr

concentrations

I I/GLUTAMATE

3 (mM)
of Fig. 2 showing data may also be

Equilibria
Over-all Reaction-The equilibrium constant of the over-all reaction, determined by chemical and enzymatic analyses of the substrate concentrations at equilibrium, is 7 i 1 (8, 9). We may, according to Equation 3 and Table I, formulate the reaction equilibrium as in Equation 6.

FIG. 4. Secondary plot from the intercepts The the extrapolations for l/V, and -l/Kc. plotted to yield Koz. TABLE II
Michaelis The 26. experiment constants and relative maximal experimental values was carried out at pH

velocities,

7.4 in 0.04

M arsenate VrPf
5/1.5

at

(6)
The term on the right is the Haldane relation for this particular mechanism (10, 11) and may be verified by substituting for the Vs and Ks the corresponding expressions in terms of rate con-

KA

K?

KG

Koz

9n.M 0.9

m.u
0.1

?nM
4

9n.u
0.04

2112

Glutamic Oxaloacetate Transaminase Mechanism

Vol. 237, No. 7

and the same data may be utilized in computing the equilibrium constants of the half-reactions. Thus we may write
KI=o(E')=-=-

(A)(E)
(G) (El

k&4
k&3

KAV, KozVr

(74

k&k;

KGVr

K1r=o(E)=kslcs=K,V,

Substitution into Equation 7 of the numerical values for the Ks and Vs from Table II gives Kr = 74 and Krr = 12. The ratio of these K values is 74/12 = 6.2, which is merely a restatement of Equation 6. In both of the half-reactions, the equilibria favor the formation of aldehyde enzyme and free amino acid, and this tendency is more marked with the aspartate than with the glutamate system. By virtue of the carboxyl groups both cr and fl to the keto function, oxaloacetate is both kinetically and thermodynamically less stable than ketoglutarate, and it is

1401 5
-log

I 4
[G or A]

I 3
(molar )

8 2

.40-

FIG. 7. Spectrophotometric equilibrium titration of phosphopyridoxal enzyme with glutamate and with aspartate in 0.04 M sodium arsenate, pH 7.3. The enzyme concentration may be determined from (AA860)max with a molar absorption coefficient calculated from Fig. 6. therefore to be expected that its amination equilibrium proceed farther toward completion. The relatively large equilibrium constants are due in part to the fact that the aldehyde group of pyridoxal phosphate in its enzyme complex is not free but occurs in Schiff base linkage with the e-amino group of an adjacent lysine side chain (12). The tendency of the coenzyme to form this internal bond serves to promote the elimination of substrate in the amino acid form from the Schiff base enzyme-substrate intermediate. The enzyme-coenzyme complex thus has a somewhat smaller amino group-accepting potential than do the amino acid-keto acid pairs. SpectrophotometricTitration-Bound pyridoxal phosphate exhibits an absorption maximum at 362 rnp in neutral and alkaline solvents, and the band shifts to 333 rnp upon conversion to the pyridoxamine phosphate form. The spectral changes with successive additions of aspartate to the aldehyde enzyme are illustrated in Fig. 5. Titration curves of the amino and aldehyde forms of the enzyme with keto and amino acid, respectively, are shown in Figs. 6 and 7, where absorbancy changes at 362 mp are used as the indicator. As expected from the kinetic analysis of the half-reaction equilibria, a large molar excess of amino acid is needed for complete conversion of aldehyde to amino enzyme, and glutamate is a better amino donor than aspartate. In the reverse titrations, the conversion of amino to aldehyde enzyme proceeds nearly stoichiometrically with additions of keto acid at micromolar concentrations. There is little question concerning the qualitative interpretation of these results. They represent predominantly the equilibria of the half-reactions corresponding to Equations 7a and 7b. A quantitative interpretation requires the proper assignment of molecular species on the basis of the absorption changes observed. In particular, the possibility must be considered that the apparent equilibria are shifted by the accumulation in significant amounts of enzyme-substrate intermediates. Since the spectra of the amino and aldehyde enzymes are not markedly altered by dialysis, we may as a first approximation assume that intermediates do not accumulate, although we cannot exclude the possibility that they occur in forms that are not spectrally distinguished

$ .305 g .20cn : .lO-

320 340 360 380 WAVELENGTH (rnp) FIG. 5. Spectral changes in the conversion of the transaminase from the pyridoxal to the pyridoxamine phosphate form by titration with aspartate.

KETOGLUTARATE,

micromolar

FIG. 6. Spectrophotometric equilibrium titration of the transaminase in the pyridoxamine phosphate form with ketoglutarate in 0.04 M sodium arsenate, pH 7.3, at 26. The intersection of the extrapolated initial slope with the limiting absorbancy gives the concentration of the bound coenzyme.

July

1962

X. F. Velick

and J. Vavra
7v, ASPARTATE (mM) 0.4 a,b,c,d 1.6 e,f,g

2113

from those of the free enzyme coenzyme complex. On the assumption of no accumulated substrate complex, the reactions considered are described by Equation 2, and the equilibria are those of the half-reactions (Equation 7). We find from the mid-points of the curves of Fig. 7 and the known concentrations of added amino acid that the aspartate oxaloacetate enzyme equilibrium constant is K1 = 150 and that the glutamate ketoglutarate enzyme equilibrium constant is KII = 40. Both values are larger than the corresponding numbers obtained kinetically, and the ratio, KI/KII = 3.7, is less than half of the correct value. The discrepancy might be attributed to the accumulation of amino acid enzyme complexes, since the amino acid concentrations exceeded 1 mM. However, the value of KII computed from Fig. 6, where the ketoglutarate concentration and the formed glutamate were in the micromolar range, is also in the vicinity of 40. Titrations of the amino enzyme with oxaloacetate give values of K1 between 80 and 200 and are difficult to control precisely. The discrepancies are not quantitatively accounted for at the present time. Znhibition Substrate Analogues-The transaminase is inhibited by substrate analogues that are incapable of undergoing transamination. Several such compounds, all aliphatic dicarboxylic acids, have been examined by Jenkins, Yphantis, and Sizer (4), who found that those compounds that were inhibitory at one arbitrary concentration also formed an enzyme complex in which the pH dependence of the absorption spectrum of the bound pyridoxal phosphate was altered. In this section, we derive the rate equations for the inhibited systems, establish experimentally that the substrate analogues are competitive with all four substrates, derive the dissociation constants of the enzyme-inhibitor complexes both kinetically and by spectrophotometric equilibrium titration, and show that the substrate analogues do not discriminate between the pyridoxal and pyridoxamine phosphate forms of the enzyme-coenzyme complex. Rate Equation for Competitive Znhibition-If a competitive inhibitor acts by occupying a site on the enzyme that is utilized singly by all four substrates, then only two inhibited complexes need be considered, namely, those formed by the inhibitor with the amino and aldehyde forms of the enzyme-coenzyme complex, respectively. The numerical indices of the rate constants of the equilibria of Equation 8 are continued in sequence from those of the minimal mechanism (Equation 4). E+IThe initial velocity kg km EI and E + I -$ EI 9. (8)

4-

3-

-2

-I

I
MALEATE

3
(mM)

--e

FIG. 8. Plot of the data for maleate inhibition of the aspartate ketoglutarate transamination to yield, at the intersection point, the dissociation constant of the complex between maleate and the pyridoxal phosphate form of the enzyme.

at a series of aspartate concentrations are shown in Fig. 8. A set of straight lines is obtained that intersect in the upper lefthand quadrant. One may solve Equation 9 for the intersection point, projected on the (-I) axis. It is found to occur at which, from Equation 8, is the dissociation C-0 = holks, constant of the complex between maleate and aldehyde enzyme. This constant is found to be 2.3 mM. A corresponding plot of 1 /Vi against (I) at a series of fixed concentrations of ketoglutarate and an arbitrary fixed concentration of aspartate also yields a set of lines that intersect in a left-hand quadrant (Fig. 9). It may be shown that the intersection point in this case is (-I) = &/i&, which, according to Equation 8, is the dissociation constant of the complex between maleate and the pyridoxamine form of the enzyme. In this case also, the intersection occurs at (-I) = 2.3 mM and is independent of the arbitrary fixed concentration of A. The combination of maleate with enzyme is thus independent of the form of the coenzyme and is determined exclusively by the specificity of the protein. Similar results are obtained when the inhibition is studied with glutamate and oxaloacetate as substrates. These observations provide additional support for the idea that all four substrates utilize the same catalytic site on the protein. Equation 9 may also be solved for the intersection point projected on the l/vi axis. Thus, by substituting -i&/kg for (I) in Equation 9, we obtain Equation 10.

b.

KETOGLUTARATE 0.08mM J.

ASP CmM)

o--o

0.64mM 0. 4 /-I

expression takes the form of Equation

In a plot of l/vi against l/(A) or l/(a) at a series of inhibitor concentrations, the inhibitor affects both the slope and the intercept, and the lines will not intersect on the ordinate. Thus, although in this case the inhibition may be strictly competitive, the usual simple criterion of competitive inhibition is lacking. The dissociation constants of the enzyme-inhibitor complexes are evaluated by plotting l/v against (I) at a series of fixed concentrations of one substrate and a single arbitrary concentration of the second substrate. Such graphs for maleate inhibition

MALEATE

(mM)

FIG. 9. The kinetics of the maleate inhibition ketoglutarate transamination, plotted to yield, tion point, the dissociation constant of the maleate and the pyridoxamine phosphate form

of the aspartate at the intersec-

complex
of the

between
enzyme.

2114

Glutamic

Oxaloacetate

Transaminase

Mechanism
TABLE III

Vol.

237, Xo. 7

Dissociation The value of l/vi at the intersection point may thus be either greater or less than the reciprocal of the maximal velocity, depending on the ratio of the dissociation constants in the last In the special case that the inhibitor comterm on the right. plexes of the two forms of the enzyme exhibit equal dissociation constants, the factor in brackets equals unity, and l/vi = l/Vr. This was actually observed in Fig. 8, where the intersection point is independent of the concentrations of both substrates, and is in accord with the fact that the two dissociation constants are equal. Moreover, the value of Vf obtained from Fig. 9 is the same as that obtained by the method of Fig. 3 in the absence of inhibitor. This unusual way of obtaining a maximal velocity is restricted to kinetic mechanisms of the transaminase type. Xpectrophotornetric Titration of Enzyme with Maleate-An independent value for the enzyme-maleate dissociation constant may be obtained by an equilibrium titration of the enzyme. At pH 7.4, the pH of the kinetic experiments, the enzyme-pyridoxal phosphate complex exhibits a strong 362 rnp absorption maximum arising from the alkaline form of the bound pyridoxal phosphate and a weak maximum at 430 rnp arising from a small equilibrium concentration of the acid form. Formation of the maleate enzyme complex raises the pK of the hydrogen ion dissociation that controls the spectrum of the bound coenzyme and hence causes a weakening of the 362 rnp band and a strengthThe concentration of the complex is ening of the 430 rnp band. proportional to the magnitude of the spectral change. Absorbancy at 360 rnp plotted against the log maleate concentration gives a typical sigmoid dissociation curve (Fig. 10). Since inhibitor concentration is greatly in excess of enzyme concentration, the dissociation constant of the complex is given by the maleate concentration at the inflection point. The number obtained is 2.3 mM, in close agreement with the result of the kinetic analysis. A sensitive spectral indicator is not available for a similar equilibrium titration of the pyridoxamine form of the enzyme, but in view of the above results we would expect the same type of agreement with the kinetic analysis. Xpeci$city of Inhibitor-Dissociation constants of enzymeinhibitor complexes for a number of substrate analogues have been determined by the methods described above and are listed in Table III. In all cases, the inhibitors behave like maleate in that they are competitive with both the amino and keto acid substrates and interact identically with the amino and aldehyde
The 26. experiment

constants of inhibitor complexes between transaminase and competitive substrate analogues

was carried out at pH 7.4 in 0.04
M

arsenate

at

Inhibitor

Succinate........................... Maleate............................. Fumarate........ Glutarate........................... Adipate................ Pimelate.......... o-Phthalate......................... m-Phthalate p-Phthalate ,.........._

18 2.3 .._.

Large
3 5

Large
5 8 10

I:..::::::::

forms of the enzyme-coenzyme complex. In order to study the kinetics of the inhibition by the aromatic dicarboxylic acids, it was necessary, because of optical interference in the 260 to 280 rnp region, to measure oxaloacetate production in a coupled enzyme system with an excess of malic dehydrogenase and reduced pyridine nucleotide. Although the phthalate isomers are also substrate analogues for the malic dehydrogenase, they did not inhibit this enzyme in the concentration region employed. It is clear from the results that the protein-inhibitor interaction is strongly influenced by planarity, intercarboxyl group distance, flexibility and other factors. There is a greater conformational in the Cs and Cg acids than in succinate, and they are bound Pimelate is flexible, but the fold in the methylene more strongly. chain is presumably too bulky when the carboxyl groups are in the favored orientation for binding. It is not possible to make any easy correlation between the behavior of the aliphatic and aromatic series of inhibitors. Inhibition by Hydroxyylamine-This reagent differs from the previously described inhibitors in that it should have no stereospecific interaction with the protein portion of the substratebinding site of the enzyme but should react exclusively with the aldehyde form of the bound coenzyme. If the enzyme-inhibitor compound is formed reversibly, then the rate equation for transamination in the presence of hydroxylamine should be obtained from Equation 9 by striking out the term containing kn/klz, which becomes zero since no amino enzyme-inhibitor complex would be formed (Equation 11). _ _ U)KAh EO
Vi Vr(A)ho +Ff

1 (
l+o+o

Ka

Kc,
>

(11)

I I 2 3 LOG [MALEAT~J

I 4 mM

I 5

FIG. 10. Spectrophotometric equilibrium titration of the The pyridoxal phosphate form of the transaminase with maleate. dissociation constant of the complex, given by the maleate concentration at the mid-point, is the same as the values determined kinetically.

If, as in the case of the substrate analogues, one plots 1 /vi against the inhibitor concentration, (I), at a fixed value of OLand a series of fixed concentrations of A, then a set of lines should be obtained that intersect in the upper left-hand quadrant. As before, the intersection point projected on the abscissa is ( -I) = klo/lcs, the dissociation constant of the enzyme-hydroxylamine compound. The experimental data so plotted are shown in Fig. 11Z. The apparent dissociation constant is unexpectedly small, of the order of 10 PM. When the complementary set of data is plotted, l/vi against (I) at an arbitrary fixed concentration of A and a series of concentrations of OL,then a set of parallel lines should be obtained,

July

1962

S. F. Veliclc and J. Vavra analogues, the individual substrates should bind both to the amino and aldehyde forms of the enzyme-coenzyme complex. In addition to the reactive complexes, one should also obtain, at suitably high concentrations, the abortive or nonreactive complexes of the type, keto acid-aldehyde enzyme and amino acid-amino enzyme. Formation of such complexes should result in inhibitions that are competitively overcome by the complementary substrates. Inhibitions of this type were not observed in the double reciprocal plots of Figs. 1 and 2 because the concentration range covered was too low, but they are observed in Fig. 14, where the substrate concentrations are elevated to levels of the order of 10 x Ks. The competitive nature of the effect is indicated by the fact that the concentration of the first or independently varied substrate at which the upward inflection of the curves occurs increases as the concentration of the second or fixed substrate is increased. Although the analysis is rather cumbersome, it can be shown that the inhibition constants of amino and keto acids are much more nearly equal than their respective Michaelis constants. The dissociation constants of the abortive complexes, determined by spectrophoto-

A= 4.0mM

- .Ol

0 NH20H

.Oi hM)

.02

,005

,010
N&OH

,015
(mM)

,020

FIG. 11. Inhibition of the transaminase by hydroxylamine. I, Competition of hydroxylamine with aspartate for the bound pyridoxal phosphate of the enzyme at 0.5 mM ketoglutarate, pH

7.3. The intersection point, projected on theabscissa, corresponds to a dissociation constant of 10 PM for the enzyme-inhibitor complex. II, The noncompetitive inhibition of the enzyme by hy-

droxylamine

with respect to ketoglutarate

concentration.

one line for each value of (Y. As shown in Fig. llZZ, this is the result actually observed. We therefore conclude from the kinetic analysis that the primary inhibition by hydroxylamine occurs, as expected, by reaction with the aldehyde group of the bound coenzyme and that the enzyme in the amino form is inert to the reagent. A spectrophotometric analysis of the effect follows. Xpectrophotometric Analysis of Enzyme-Hydroxylamine Compound-Fig. 12 illustrates the spectral shift that occurs when hydroxylamine is added to the phosphopyridoxal form of the enzyme. The absorption bands of both the acid and alkaline forms of the bound coenzyme, pH 5.5 and 8.5, shift to the region of 380 mp, corresponding to the formation of the hydroxylamine adduct. In accord with the kinetic analysis, 50% conversion occurs at hydroxylamine concentrations in the range from 10 to 20 PM. Also in accord with kinetic analysis, the spectrum of the phosphopyridoxamine enzyme is unaffected by hydroxylamine. It is necessary for such a test to dialyze the amino enzyme free from any traces of keto acid. If, for example, in the presence of stoichiometric concentrations of ketoglutarate, the enzyme is maintained predominantly in the amino form by a high concentration of glutamate, the addition of hydroxylamine in the millimolar concentration range suffices to pull the equilibrium toward the aldehyde form by formation of the hydroxylamine aldehyde adduct. This is illustrated in Fig. 13. The apparent conversion of amino enzyme to aldehyde enzyme hydroxylamine adduct is mediated by the trace of ketoglutarate formed in the original conversion of aldehyde to amino enzyme and proceeds to an extent that is limited by the amount of ketoglutarate present. The formation of the hydroxylamine enzyme adduct is reversible. Thus, if the dialyzed aldehyde enzyme in the presence of 10 to 20 PM hydroxylamine is treated with glutamate at a final
concentration declines and of 10 mM or higher, the 380 rnp absorption band

or

360

400 320 360 WAVELENGTH,

400 my

440

460

FIG. 12. Spectra of the bound pyridoxal nresence and absence of hydroxylamine. 5.5.

phosphate

in

the

I, At pH 8.5; 11, at pH

a 0 t

I I I I I I I I I I I 420 380 340 WAVELENGTH, m/c

Therefore,

the 335 rnp band of the amino enzyme appears. as indicated by the kinetic analysis, the enzyme

inhibitor compound is dissociable. The hydroxylamine reaction with the bound coenzyme presumably occurs by displacing the internal lysine bond and is thus expected to go directly to the oxime. In the nonenzymatic case, oxime formation involves addition followed by elimination of the elements of water and proceeds toward completion only at much higher concentrations

of reactants. Inhibition by Excess Substrate-As

in the case of the substrate

FIG. 13. Curve 1, absorption spectrum of a dialyzed mixture of the amino and aldehyde forms of the transaminase. Curve 2, absorption spectrum of the same mixture after addition of excess glutamate showing ostensibly complete conversion to the amino form. Ketoglutarate is now present at a concentration equal to that of the aldehyde enzyme, which has disappeared. Curve 3, effect of hydroxylamine addition to the system of Curve 2. By combining with the extremely low equilibrium concentration of aldehyde enzyme, the inhibitor has pulled the amino and aldehyde enzyme concentrations back to the original level limited by the amount of ketoglutarate formed in the original conversion. In the absence of ketoglutarate, the amino enzyme is unaffected spectrally by hydroxylamine.

2116

Glutamic Oxaloacetate Transaminase Mechanism

Vol. 237, No. 7

I 2 I/KETOGLUTARATEhnM)

FIG. 14. Substrate inhibition by ketoglutarate as a function of aspartate concentration at pH 7.3. Inhibition, as indicated by the upward inflection of the curves, begins at higher ketoglutarate concent,rations as the aspartate concentration is increased. metric equilibrium titration, are utilized in a later section in the evaluation of individual rate constants. They are considered also in the study of the pH dependence of maximal velocities, since their pH dependence is different from the pH dependence of the reaction steps and cause artifacts which must be controlled. Aspects of substrate and product inhibition are utilized for other purposes in the following section, which treats the behavior of the so-called three-substrate systems.

These equations, which correspond to the four three-substrate cases, are all of the same form, and examination of them reveals that equivalent information may be obtained by the analysis of either of two pairs, Equations 12a and 13a or Equations 14~ and 15a. In order to condense the equations, we have substituted for the appropriate aggregates of individual rate constants the symbols for the equivalent Michaelis constants and maximal velocities, leaving in explicit form only those rate constants or ratios thereof to be evaluated experimentally. Experimental Procedure-The experimental results are treated by two successive graphical analyses. With Ox-G-o! system (Equation 14a) as an example, ai is measured as a function of the inhibitor concentration, (a!), at fixed concentrations of 0% and G. A plot of l/vi against (a) yields a straight line. With (G) kept constant, the measurements are repeated at a series of fixed concentrations of Ox. Plots of these results yield a series of straight lines, one for each concentration of Ox, and the lines are found to intersect in a left-hand quadrant. Several similar series of measurements are made at different fixed concentrations of G. The intersection points, projected on the abscissa or (o() axis, are functions of (G). Two such sets are illustrated in Fig. 15. The data for the other three-substrate cases are obtained and treated in the same way with similar results. One may solve Equations 12a to 15a for the intersection points obtained in graphs of the above type. The relations are expressed in reciprocal form, with the inhibitory product in each case written on the left (Equations 12Z1to 15b).

Three-substrate Systems
Theory-An independent kinetic determination of the equilibrium constants of the half-reactions and a further separation of the individual rate constants is obtained by analysis of initial velocities measured in the presence of one reaction product. There are four three-substrate cases that may be examined, corresponding to the substrate combinations, A-or-G, G-Ox-A, Ox-G-a, and a-A-Ox. In each instance, the inhibitor is written on the right, and the substrate with which it competes, on the left. The inhibitors actually engage in transaminations, but under initial velocity conditions they can only react with the central members of the trios by reactions in which both partners are regenerated. The net effect is withdrawal of enzyme from the reaction being directly observed and an apparent inhibition. Initial velocity equations for these systems have been derived for the steady-state condition and for both the minimal and the expanded kinetic mechanisms. As before, expansion of the mechanism does not alter the form of the results, and we therefore present the equations for the minimal mechanism involving only one explicit intermediate per half-reaction.

7J * I
4 3

~LIJ mM 0.4

CURVE 0 b c e f

[OXALACJ flbM 0.02 0.04 0.12 0.02 0.04 0. I2

6.4

I
I. 1 -0.3 I - 0.2 I -0.1 0 [KETOGLUTARATE] I I 0.1 mu I 0.2 I OS3

FIG. 15. Inhibition of the glutamate oxaloacetate reaction at pH 7.3 by ketoglutarate. Reciprocal initial velocities are plotted against ketoglutarate concentration at three oxaloacetate concentrations, each at two different concentrations of glutamate, according to Equation 14~. I q
3OXALACETATE 0.06mM /A/ GLUmM
l

Wa)
(13a) 1 -=vi I -=Vi

b)
vr (OS) Vf

(02)

Koz 4
i 6

ksh

E (G) 6 8

(14a)

I3 -1.2

I -0.8

I I 89 -0.4 0 ASPARTATE

I ! 9 0.4 0.8 (mM)

I 1.2

(15a)

FIG. 16. Inhibition of the glutamate oxaloacetate transamination by aspartate at pH 7.3, plotted according to Equation 13a.

July

1962

X. F. Velick and J. Vavra

2117

All of the three substrate cases have been studied, and the results are summarized in Table IV. The equilibrium constants of the half-reactions, obtained by this method, are in essential agreement with the results calculated from the Michaelis WI constants and maximal velocities of the two substrate systems. The apparent dissociation constants, k&r, etc., are further (14b) analyzed in the following section. Dissociation

Constants

(15b)
Thus, for the case we are examining in detail, Equations 14~ and 14b, a plot of the reciprocal of the intersection points, - (1 /cr) a, against the corresponding reciprocal concentration of G gives a straight line (Fig. 17)) the slope of which is k&,/i&&, the equilibrium constant of the glutamate half-reaction, and the intercept From these quantities one of which on the ordinate is k5/&. may calculate k,/Jcs. The latter ratios are, respectively, the equilibrium constant for formation of intermediate from amino enzyme and ketoglutarate and the equilibrium constant for the dissociation of intermediate to form aldehyde enzyme and glutamate.

-75 j i
0.6

Expanded Mechanism-The theoretical treatment of the kinetic experiments in the preceding sections was based upon the minimal mechanism, both for algebraic simplicity and because additional constants could not be resolved solely by the Insofar as the over-all equilibria methods of over-all kinetics. and the equilibria of the half-reactions are concerned, the minimal mechanism introduces no ambiguity. On the other hand, the apparent dissociation constants of the enzyme-substrate complexes, as defined by the minimal mechanism and listed in Table IV, are not microscopic constants but involve intramolecular reactions in addition to the association and dissociation processes. A more detailed analysis of the product inhibition experiments corresponding to Fig. 17 and Table IV requires consideration of For this purpose, a partial an expanded kinetic mechanism. expansion to two intermediates per half-reaction will suffice (Equation 16).
A+EyEA

&
kz

ka
k

ks
EOx e kg E + Ox (16a)

kGLUTAMATE

(m M 1

IO 20 bOXALACETATE

30 (mM)

Left, reciprocal intersection points, with respect to the abscissa, for the system of Fig. 15, plotted against the corresponding reciprocal glutamate concentrations (Equation 14b). Right, reciprocal intersection points for the system of Fig. 16 plotted against the corresponding reciprocal concentrations of oxaloacetate. The intercepts on the ordinates are, respectively, ka/ka and kl/kp, and the slopes are as indicated.
FIG. 17.

The rate constants for the expanded mechanism are in bold face type to distinguish them from those of the minimal mechanism. Since there are, in fact, at least twice as many intermediates as those formulated in Equation 16, it may appear arbitrary to designate the two intermediates per half-reaction explicitly as enzyme-substrate and enzyme-product complexes. Justification for this procedure is provided in the following paragraph.
IV in three-substrate systems

TABLE Intermediate All equilibria

from

kinetic
M

analysis

of competition

measurements

are based on conditions

of 0.04

sodium arsenate, pH 7.3, 25.

I 0x-G-a G-0x-A I a-A-Ox

A-a-G

Equation Half-reaction
From From Apparent

12b

14b

136

15b

equilibrium

slope or its reciprocal K, and v,,, (Equation association constant

8.8
7)

I
12 ks -=ks

11.4

55

I 74 EX kd -=b

72

ks -=__
h above) 0.2 0.15 slope of the paired

EY G.E

EY ol.E 1.7 0.6

h -=kz 0.5 0.38 are within

EX 0x.E 28 28

A-E

From intercept Calculated* * Calculated from secondary plots.

(equation

I errors

intercept

and

system.

The

discrepancies

the probable

of the primary

and

2118

Glutamic Oxaloacetate Transaminase Mechanism

4 -x--c k3

Vol. 237, h-o. 7

k, k, - ho

The rate equations corresponding to Equation 16 are identical with Equation 4, but the expressions for the Michaelis constants and maximal velocities in terms of individual rate constants are more complicated (Table V). Similarly, the equations for the three-substrate systems (Equations 12 to 15) are enlarged but retain the same form. We will not present the expansion of these equations in full, but the reinterpretation of the slopes and intercepts of the secondary plots corresponding to Equations 12b to 15b and Fig. 17 are summarized in Table VI. It may be seen that the slopes are still the equilibrium constants of the half-reactions, but the intercepts now contain two terms, which may be factored. In each case, the factor outside the parentheses is the equilibrium constant for the initial or terminal step
in a half-reaction. The same type of factored intercept is ob-

b \,,,s5 I ,, ..../ 2:,,_!..\.*, ,

(1SSOC xiz + dlsplocement -displacement +H+ -H+ - -H+ +H+ + H,O 5-c 2

-_ 4-.,:: i% : \ ...,,I .,,, 2:,I*I. ._ I. 0

dissoc. assoc.

FIG. 19. Diagram four intermediates

of the showing

mechanism of a half-reaction the enzyme-substrate and

with enzyme-

product complexes and the two isomeric Schiff base intermediates.

Superposition of substrate and bound coenzyme shows one way in which the proximity effect of a substrate or substrate analogue carboxyl group could influence the spectrally linked pK of the coenzyme. The amino group of the substrate and the e-amino group of the adjacent lysine side chain competitively displace each other from azomethine bonds to the carbonyl group of the coenzyme. On the other side of the sequence, the isomeric Schiff base is reversibly formed and cleaved by the elimination and addition of the elements of water. As the figure is drawn, the amino group of the lysine and the phenolic group of the coenzyme could catalyze the hydrogen shift in the isomerization of the intermediate, but the results and arguments summarized in Numerical Evaluation of Individual Rate Constants provide no direct evidence for such an interpretation.

tained

regardless

of the number
TABLE

of additional
V

intermediates

Michaelis rate K A_ Vr(kzks

constants and maximal velocities in terms constants of partially expanded mechanism + ksks W&s + knkr) K a = Vr(k&lo f k&l k?kokn

of

+ k&l)

= ksklo(kz

+ k,

kzkakskm + k4) + kekr(ks

+ ks + kd

TABLE Interpretation (Table IV)

T

VI of three-substrate kinetics expanded mechanism

G-0x-A I a-A-Ox

of secondary plots in terms of partially

A-a-G 0x-G-a

Slope

Intercept

--1-

FIG.

18.

phosphate
concentrations tion constants

Spectrophotometric equilibrium titration of pyridoxal enzyme with ketoglutarate and oxaloacetate. The
of the keto acids at the mid-points of the respective abortive complexes, are the dissociaE-a and E-Ox.

postulated in the kinetic mechanism. In the general case, the factor outside the parentheses is always the equilibrium constant of an initial or final step, and the constants within the parentheses correspond exclusively to intramolecular reactions within the complex. Analysis of over-all kinetics does not enable us to separate the bimolecular from the intramolecular factors, but if the pure association or dissociation constants of two of the enzyme-substrate complexes can be determined by independent means, then the corresponding constants for the other two complexes may be computed, and in addition one may obtain the steady state equilibrium ratios of the two macroscopic intramolecular rate constants of each half-reaction, namely, ks/klo and k4/ks. It will be shown that this can be done, at least to a first approximation, and that the results taken together with other kinetic information permit the numerical evaluation of k3, k4, kg, and km Approximation of Dissociation Constants-The two enzymesubstrate dissociation constants that may be determined experimentally are those of the nonreactive complexes between phosphopyridoxal enzyme and the two keto acids. These are determined by equilibrium titrations, which are based upon observations of the same spectral shifts that are produced in the complexes between enzyme and the competitive substrate analogues such as maleate. The two titration curves are shown in Fig. 18 and yield dissociation constants of 3 and 6.7 mM for E-a, and E-Ox. In order to equate these values, respectively, with ks/k7 and k5/k6 of the expanded mechanism, it is necessary to assume that the keto acids, like the substrate analogues, are not strongly affected by the state of the functional group of the coenzymes in their initial interaction with the protein. This assumption would appear, at first, to involve neglect of the fundamental nature of the catalysis. It may be rationalized in the case of the amino acid substrates because the carbonyl group of the coenzyme is in azomethine linkage to a lysine side chain and is pulled to one side, as illustrated diagrammatically in Fig. 19. In the case of the active keto acid complex, the free

July

1962

S. F. Velick

and J. Vaura controlled rates according to the analysis of Alberty and Pierce (13). If we are dealing with rates of this order of magnitude, then IQ, kg, kg, and kll are also large, since the dissociation constants are all between 10e2 and lop3 M. Accordingly, the expressions for maximal velocity in the expanded mechanism (Table V) reduce to a simpler form (Equation 17).
k&s Vf = __ k3 + ks

amino group of the coenzyme can cause steric interference, but it also has some degree of rotational freedom, and the interference, energetically, may therefore not be large. If we accept these arguments provisionally, then multiplication of the numerical value of kg/kT, so obtained, by the experimental value of kF/ks(l + kg/km) from Table VI gives us the numerical value of the factor in parentheses from which we obtain the value of The latter number turns out to be 4.0. Substitution ks/km of the reciprocal of this number in the appropriate parenthetical term of Table VI gives us the numerical value of klz/kn, the association constant of the enzyme-glutamate complex. The same sequence of operations with ks/kc from the abortive oxaloacetate complex gives us k4/k3, the intramolecular equilibrium of the first half-reaction, and kl/kz, the association constant of the enzyme-aspartate complex. The numbers so obtained are listed in Table VII. Numerical Evaluation
of Individual Rate Constants

and

v, = 1~ + ho

lcakm

(17)

These relations, together with the equilibria of the type, kI/k2, etc., of Table IV complete the number of independent relations required for the algebraic solution of the intramolecular rate constants of the partially expanded mechanism. Thus, if kr/ks = a and ks/klo = b, it may be shown (Equation 18) that
k 3 = Cab Wrb -

l)VfV,
Vfh

and

klo =

(1 (Vr -

ab)VfV,
aVr)b

(18)

The analysis up to this point yields simple ratios of rate constants. Those ratios corresponding to initial or final steps of the half-reactions are clearly defined as association and dissociation equilibria, provided only that two or more intermediates are postulated per half-reaction, as in Equation 16. They remain so defined, with the numerical values in Table VII, regardless of any further expansion of the kinetic mechanism. Mechanistically they apply to those events that occur before Schiffs base formation between substrate and coenzyme or the events that occur after the cleavage of the intermediate. To what extent they correspond to elementary processes is of interest but is irrelevant to the present argument. On the other hand, the so-called intramolecular rate constants, ks, kh, kg, and klo, are definitely macroscopic constants in the sense that each describes a sequence of elementary processes. It is therefore possible to describe each of them as a function of a still larger number of specific intramolecular rate processes, but it is pointless to do so for the treatment of over-all kinetic and equilibrium data because they could not be evaluated from such information. Each macroscopic k nevertheless describes the rate of a particular intramolecular sequence in the direction indicated. Moreover, in a special case, of which the transaminase appears to be an example, they may be evaluated numerically from the data at hand. The special case is that the bimolecular steps involving the association of substrate with enzyme are not rate-limiting and are of the order of diffusion controlled values. The experimental support for the argument that kl, kg, k?, and kIz are extremely large is provided by the facts (a) that on such an assumption one may derive values of the four intramolecular ks that predict the observed rates of isotopic exchange in the isolated half-reactions and (b) that even if it is rate-limiting, kg, for example, would have to be of the order of 10 M-I see-l to account for the observed V, under the conditions at which it is attained. If the intramolecular steps were not rate-limiting, they would have to be improbably large. It is pertinent to point out that the observed results are described by the steady state approximation and that in the steady state approximation the bimolecular rate constants do not appear in the expressions for the maximal velocities. This is a general property of such mechanisms, although it is usually stated that the steady-state approximation makes no assumptions about rate constants. It is also pertinent to mention that the association of fumarase with its dicarboxylic acid substrates, which are similar to those of the transaminase, also approaches diffusion

The other two intramolecular constants, k4 and ks, may also be related to a, b, and the maximal velocities or may be obtained from kt and klo by simple relations which have already been given. Substitution of the appropriate numerical values of a and b and the maximal velocities from Table VII into Equation 18 give us the numerical values of the intramolecular rate constants listed in Table VIII. According to these results, the rate-limiting step in the transamination of ketoglutarate by aspartate occurs in the first half-reaction and is described by k3, the rate of conversion of bound aspartate and pyridoxal phosphate to bound oxaloacetate and pyridoxamine phosphate. Similarly, the rate-limiting step in the reverse reaction is described by klo, the intramolecular conversion of glutamate to ketoglutarate. The large numerical value of k4 is required by the equilibrium of the first half-reaction. Isotopic Equilibration-It is possible, on the basis of the results in Table VIII, to predict the rate of the steady state isotopic equilibration reaction (Equation 19) Glutamate-W + ketoglutarate-CY f glutamate-C* + ketoglutarate-Cl4 (19)

compared to the initial maximal velocity, Vf, of the over-all transamination. The exchange rate is given by a standard expression, Equation 20 (14), which is independent of the reaction mechanism (G) (c40.693 Vex = [(G) + (a)lt;
TABLE Dissociation
kz -=_ kg
w&M

(20)

constants
kc EJ.0.X - = @Or) kc
mu

VII of enzyme-substrate
ks E.o: - = (Ea) k7
,?LM

complexes
ku -=kn
VZM

l3.A (EA)

E.G (EG)

6.7

8.3

TABLE Rate constants for intramolecular ylutamic oxaloacetate

ka

VIII
reaction transaminase ks sec9 4170 sequences of

ka

km
MC-

seco 323

se0 59,100

1020

2120

Glutamic

O.raloacetate

Transaminase

Mechanism

Vol. 237, No. 7

where ti is the half-time for complete isotopic equilibration. If the experiment is carried out at substrate concentrations that give approximate maximal velocity, then, purely by symmetry considerations, the exchange velocity, V,,, is a maximal velocity given by the expression (Equation 21) k&m v,, = ~ ks + ho
(21)

pH Dependence of Enzyme Activity Maximal Velocity pH Dependence-With the methods described in Kinetic Parameters, the maximal velocities of the forward and reverse reactions were studied over the pH range from 5.0 to 10.0. In order to cover this range, it was necessary to establish the absence of specific ion effects from the different buffers employed, to make corrections for the changes in absorption coefficient of oxaloacetate as a function of pH, and to cope with possible irreversible or secondary changes in the more extreme pH conditions. The various complications were most troublesome above pH 9, and we have not succeeded in obtaining a reliable estimate of a rate-controlling pK in the alkaline region, although we believe one to occur in the vicinity of pH 10. The results for the forward reaction are summarized in Fig. 20 and Table IX. Difficulties were encountered in the acid region because the Michaelis constant for ketoglutarate decreases with pH, and the sensitive concentrations become too small for optimal treatment even by the expanded scale optical method. Close analysis was particularly important because deviations in double reciprocal plots due to inhibition by excess keto acid are more marked in the acid region. It was possible to check the maximal velocities and to circumvent both difficulties by making use of a relation observed in connection with maleate inhibition (Figs. 6, 7, and 8). It was shown in the latter analysis that the intersection points of l/Vi against (maleate) plots at a series of different aspartate concentrations and a single fixed ketoglutarate concentration give not only the dissociation constant of the maleate-enzyme complex but also a true, uninhibited, maximal velocity (Equation 10). The latter curves may be run at the lowest fixed ketoglutarate concentration that gives measurable initial velocities and have been utilized to check the maximal velocities at pH 5.0, 5.5, and 6.0. The results also explain the increased inhibition by ketoglutarate at acid pH, since the dissociation constant of the maleate-enzyme complex, which inhibits exactly like the abortive ketoglutarate-enzyme complex, decreases from 2.3 mM at pH 7.4 to 0.36 mM at pH 5. Attempts to measure velocity-pH functions at high fixed substrate concentrations lead to erroneously high and variable pK values in the pH activity curves because of the pH dependence of inhibition by ketoglutarate. Thus, at aspartate and ketoglutarate concentrations of 6.7 DIM, there is an apparent rate-controlling pK around pH 7 (4). Any correlation of such a pK with the protolytic pK of the abortive ketoglutarateenzyme complex is coincidental. One may, however, obtain a fair approximation of the present results by running the pHactivity curve at fixed substrate concentrations of aspartate, (20 mm) and ketoglutarate (0.5 mm). These concentrations give a good approximation of maximal velocity, and at the same time any competitive substrate inhibition is eliminated. The same type of broad pH-maximal velocity plateau is obtained for the transaminase reaction studied in the reverse direction with oxaloacetate and glutamate as substrates. However, oxaloacetate is more difficult to cope with in the acid region than is ketoglutarate, and we have not attempted to extend the analysis with this substrate. Because of the degree of symmetry of the forward and reverse reactions, the absence of these data does not seriously handicap us. pH Dependence of ka and kio--When two rate constants are in series, the over-all rate is dominated by the smaller of the two Thus the maximal velocities and pH dependence of constants. the forward and reverse reactions are determined, respectively,

Substitution into Equation 21 of the numerical values of the rate constants from Table VIII gives V,, = 800, which we may compare with Vf = 300, and which gives the theoretical ratio, V,,/Vf = 2.7. Jenkins and Sizer (6) have actually measured the rate of equilibration of glutamate and ketoglutarate and mention the fact that it occurs at approximately twice the rate of the over-all transamination of ketoglutarate by aspartate at the same concentrations of enzyme and substrate. In the light of the present work, the amino acid concentrations they employed approached saturation levels and the keto acid concentrations were in the range that produced substrate inhibition, but the latter effect should cancel out approximately in the ratio. In view of the facts that no claims were made concerning the precision of the experimental ratio, that an unspecified and uncertain absorption coefficient was involved, and that there are approximations in the theoretical ratio, the agreement between theory and experiment must be considered to be good.

PH

and Vf/K, as functions of PH. Vr is in units of turnover number per molecule of bound coenzvme. In the acid region, the buffer is acetate with Tris as the cation. In the middle and alkaline regions. the buffer is either sodium arsenate or Tris, with acetate or chloride as the cation. Results with carbonate-bicarbonate and with glycine buffers were erratic.
FIG. 20. TABLE

Plots of Vf, Vi/K*,

IX
of glutamic

pH

dependence of kinetic parameters oxaloacetic transaminase

vf/K~

PH

vf* SIG-

KA

Vi/&

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
* In units

70 185 260 300 300 310 300 320 310 330

of turnover

3.5 2.0 2.4 1.2 1.0 0.9 1.0 1.2 1.9 2.5

0.02 0.04 0.05

0.1 0.1 0.09 0.09 0.13

0.14
of bound

53 130 130 250 310 330 310 260 170 120

coenayme.

13,000 7,500 6,000 3,100 3,000 3,500 3,000 2,600 2,200

per mole

July

1962

S. F. Velick

and J. Vavra

2121

by ks and klo and by their respective pH dependencies. The rate-controlling pK of 5.1 for Vf, therefore, corresponds to the dissociation of a hydrogen ion from a group in the enzyme-aspartate complex or in one of the associated intermediates. If the pK is that of a group of the protein or coenzyme, it is not necessarily identical with the pK of the same group in the absence of substrate. A feature of a transaminase mechanism involving a Schiff base intermediate is that an isomerization is required in which the double bond of the Schiff base is moved reversibly from the substrate side to the coenzyme side of the imino nitrogen. Such a shift involves both a gain and a loss of protons from the intermediate and is enzyme-catalyzed, since it does not occur rapidly in nonenzymatic systems. If the proton reactions are promoted by specifically oriented proton-donating and -accepting groups of the enzyme, then these groups would be expected to function in the vicinity of their respective pK values, because a group that donates a proton in the first half-reaction must accept a proton in the inverted second half-reaction. One would therefore expect to observe a sharp optimum in the pH-maximal velocity curve. The absence of a sharp optimum would occur in such a mechanism only under the conditions that have been defined, namely, that the maximal velocity in each reaction direction is dominated by one of the two half-reactions. pH Dependence of kl, ke, k7, and kn-Whereas the pH dependence of the maximal velocity is determined by ionizations in the enzyme-substrate complex, a pH plot of Vmax/Ks, where Ks is a Michaelis constant, gives the pK values of dissociable groups in the enzyme itself which affect the rate constants of the rapid bimolecular association reactions between enzyme and substrate (15). Thus, if we consider the expanded mechanism of the transaminase reaction (Table V), the functions, KA/Vf and Ka/Vf, may be written in the form of Equation 20.

linked group is appreciably raised in the enzyme-substrate or enzyme-inhibitor complex (as utilized under Three-substrate Systems and Dissociation Constants), it could not be this group, as modified by substrate in the enzyme-substrate complex, which is responsible for the pK of 5.1 that controls Vf and ka. The latter pK may correspond to a carboxyl ionization of the substrate itself or of a protein side chain. Summary Glutamic oxaloacetate transaminase has been prepared in a state approaching purity. From a spectrophotometric analysis of the experimentally accessible equilibria between enzyme, bound coenzyme, substrates, and inhibitors, and by a systematic analysis of the initial velocity kinetics of the transamination in both reaction directions in the presence and absence of one product, the following information has been obtained. 1. The reaction proceeds kinetically through two mutually inverted half-reactions with exclusively binary complexes between substrate and the functionally nondissociable enzymecoenzyme complex. 2. In accord with previous chemical information, the bound coenzyme acts as an amino group carrier. 3. The four substrates are mutually competitive and occupy the single catalytic binding site on the protein one at a time, in sequence. 4. The equilibrium constants of the over-all reaction and of the two half-reactions may be derived kinetically by two independent methods. 5. The theory of competitive inhibition for the transaminase mechanism has been developed and applied to the inhibitions produced by a series of substrate analogues. 6. Dissociation constants of complexes between enzyme-coenzyme complex and inhibitory substrate analogues derived kinetically are in close agreement with the constants derived by spectrophotometric equilibrium titration. 7. Substrate analogues that cannot engage in transamination inhibit by occupying the substrate binding site on the enzymecoenzyme complex and form complexes with the same dissociation constant whether the bound coenzyme is in the amino or aldimine form. 8. The theory has been developed and established experimentally for the case of a competitive inhibitor that interacts exclusively with one form of the bound coenzyme. Hydroxylamine reacts reversibly only with the bound pyridoxal phosphate and not with the pyridoxamine, and it competes only with the amino acid forms of the substrate. 9. By the appropriate combination of kinetic and equilibrium measurements, it is possible, for the mechanism derived, to separate the rate constants for the formation and dissociation of the enzyme-substrate complexes from those that occur intramolecularly on the enzyme and involve the formation, isomerization, and cleavage of the Schiffs base intermediates. 10. Dissociation constants of the enzyme-substrate complexes are derived kinetically. 11. The bimolecular association reactions between enzyme and substrate are not rate-limiting. The possibility that they approach diffusion controlled rates is indicated. 12. Macroscopic rate constants for the four intramolecular reaction sequences, two in each reaction direction, are derived algebraically and are evaluated numerically from the experi-

and

In both cases, the rate constants within the brackets occur in ratios, and each ratio involves a pair of constants that apply to the same intermediate. A derivation of the pH dependence of K,/Vf involves multiplying each rate constant by its own pH function, which is determined by the pK values of the intermediate. These pH functions cancel out in pairs, leaving only the pH function of the unpaired rate constant outside the brackets. This argument also applies to expanded cases with any additional number of explicit intermediates. Thus a plot of Vf/Ka against pH describes the pH dependence of kl, and a plot of Vr/K, describes the pH dependence of kl. The corresponding plots are shown in Fig. 20. The curve, Vi/K* against pH, exhibits two inflections, one at pH 6.3 and the second at pH 9. The pK of 6.3 is close to the pK of the ionizing group, which affects the spectrum of the bound coenzyme in the absence of substrate and could, for example, correspond to the ionization of the phenolic hydroxyl or ring nitrogen of the bound coenzyme. This is in accord with the theory outlined above that such a plot should reveal the pK of a group in the enzyme or enzyme-coenzyme complex and not in the enzyme-substrate complex. Since the pK of the spectrally

2122

Glutamic Oxaloacetate Transaminase

Mechanism

Vol. 237, No. 7

mental data for the favored case, that in which the bimolecular steps are very rapid compared with the intramolecular steps. 13. The constants so obtained may be used to compute the rates of isotopic equilibration in isolated half-reactions and are in accord with data in the literature. 14. The rate-limiting step in the transamination of ketoglutarate by aspartate is intramolecular and occurs in the halfreaction in which phosphopyridoxal enzyme and aspartate are converted to phosphopyridoxamine enzyme and oxaloacetate. The rate-limiting step in the reverse reaction is also intramolecular and occurs in the half-reaction in which glutamate is converted to ketoglutarate. 15. The pH dependence of the maximal velocity has been investigated in both reaction directions. There is no dependence in the region pH 6 to 9. 16. In the aspartate ketoglutarate reaction, there is a ratecontrolling hydrogen ion dissociation with a pK of 5.1. This pK corresponds to an ionization in the reactive intermediate formed by enzyme-coenzyme with the 4-carbon substrate. 17. The rate of the bimolecular association reaction between aspartate and enzyme is controlled by a hydrogen ion dissociation of pH 6.3. This pK coincides with the spectrally linked pK of the bound pyridoxal phosphate. It does not affect the maximal velocity and is involved in the reaction rate at submaximal substrate concentrations only through its influence on Michaelis constants. 18. The rate equations for the mechanisms described above were derived on the assumption that the enzyme species are in a

steady state during initial velocity measurements. Conditions are defined for obtaining the kinetic and equilibrium parameters from primary and secondary plots of the experimental data. REFERENCES
1. SNELL, E. E., J. Biol. Chem., 164, 313 (1944). 2. METZLER. D. E.. IKAWA. M.. AND SNELL. E. E.. J. Am. Chem. Sot., 78, 648 h954). 3. MEISTER, A., SOBER, H., AND PETERSON, E. A., J. Biol. Chem., 206, 89 (1954). 4. JENKINS, W. T.. YPHANTIS, D. A., AND SIZER, I. W., J. Biol. Chem.,234, 5i (1958). 5. NISONOFF. A.. BARNES. F. W.. JR.. ENNS. T.. AND VON SUCHING, S.,Buk Johns Hopkins Hdsp., 94, ll? (1954). 6. JENKINS, W. T., AND SIZER, I. W., J. Biol. Chem., 234,1179

(1959).
7. KING. E. L.. AND ALTMAN. C., J. Phys. Chem., 60, 1375 (1956). 8. KR&, H. k., Biochem. j., 54, 82 (1953). 9. NISONOFF. A.. BARNES. F. W.. JR.. AND ENNS. T.. J. Biol. Chem., 204, 957 (1953). 10. HALDANE, J. B. S., Enzymes, Longmans, Green and Company,
I / , I

Ltd., London, 1930. 11. ALBERTY, R. A., J. Am. Chem. Sot., 75, 1928 (1953). 12. FISCHER, E. H., AND KREBS, E. G., Abstracts of the 136th Meeting of the American Chemical Society, Atlantic City,
September 1959, p. 24~.

13. ALBERTY, R. A., AND PIERCE, W. H., J. Am. Chem. Sot., 79, 1526 (1957). 14. WAHL, A. H., AND BONNER, N. A., Radioactivity applied to chemistry, John Wiley and Sons, Inc., New York, 1951. 15. FRIEDEN, C., AND ALBERTY, R. A., J. Biol. Chem., 212, 859 (1955).