THE JOURNAL or BIOLOGICAL CHEMISTRY Vol. 242, No. 1, Issue of January 10, PP. 101-108, 1967

Printed in U.S.A.

Alanine Aminotransferase

II. THE BASIS FOR SUBSTRATE SPECIFICITY*

(Received for publication, June 28, 1966)

MILTON H. SAIER, JR., AND W. TERRY JENKINSI

From the Department of Biochemistry, University of California, Berkeley, California 94?‘20

SUMMARY

The substrate specificity of soluble alanine aminotrans- ferase from pig heart was examined. On the basis of com- petitive inhibition studies it was concluded that, in many cases, the low reactivities of enzyme-substrate analogue complexes were due to the effect of the amino acid side chains on the maximum reaction velocity rather than to a change in the Michaelis constant.

Formate was found to stimulate alanine aminotransferase- catalyzed amino transfer from alanine to either pyruvate or a-ketoglutarate, but to inhibit that from glutamate to either cY-ketoglutarate or pyruvate. Rates of removal of the cr-hy- drogen atoms of glutamate and alanine in DzO were affected by formate in the same manner as were the rates of transami- nation. Other monocarboxylate anions inhibited both transamination reactions. Formate acted as a noncompeti- tive inhibitor of cr-aminobutyrate transamination. Results were interpreted to mean that activation by formate was due to its binding to the site which normally binds the y-car-boxy1 group of glutamate.

No effect of formate on the spectrum of the enzyme in the presence of an excess of alanine and pyruvate was observed, and its effect on the spectrum of the enzyme in the presence of excess glutamate and cy-ketoglutarate was slight.

Two other enzymes, n-alanine aminotransferase from BaciZlus subtilis and aspartate aminotransferase from pig heart, catalyze amino transfer from alanine to ar-ketogluta- rate, at rates which are increased by monocarboxylate anions.

An evaluation of the available substrate specificity data for numerous enzymes shows that parts of a substrate molecule, even those which are distant from the site at which the enzyme- catalyzed chemical reaction occurs, influence the reactivity of the enzyme-substrate complex. A substrate analogue, which differs from the natural substrate of the enzyme only with respect to a deletion of such a distal group, usually reacts more slowly. The maximum velocity at which such a substrate analogue reacts

* This work was supported in part by Grant 5 ROl HE04417 from the United States Public Health Service.

$ Present address, Department of Chemistry, University of Indiana, Bloomington, Indiana 47401.

might be increased toward that at which the natural substrate of the enzyme reacts by introduction of a molecule which re- sembles the missing moiety into the enzymic site which normally binds this group.

In recent years several examples of rate enhancement of en- zymic reactions coincident with this expectation have been re- ported, although few have been well characterized (l-6). Nota- ble is the acceleration of the tryptic hydrolysis of acetylglycine ethyl ester by alkylammonium ions (5). The fact that binding constants of the different cations as activators of the hydrolysis of acetylglycine ethyl ester were correlated with the effectiveness of these ions in inhibiting the hydrolysis of benzoyl arginine ethyl ester, a much better substrate, indicated that activation was due to the binding of the alkylammonium ions to that part of the enzyme which normally binds the arginine side chain. In all similar cases of activation which have been reported, the reaction has involved a substrate analogue for which even the accelerated rate is only a small fraction of that with the natural substrate.

Substrate specificity requirements of soluble alanine amino- transferase (L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) from pig heart have not been well defined. Green, Leloir, and Nocito (7) demonstrated that cr-aminobutyrate (replacing alanine) and mesoxalate (replacing pyruvate) were transaminated by their enzyme preparation, but neither the relative rates of transamination nor the specific activity of the enzyme preparation used was reported. In the present investi- gation, the reactivities of various enzyme-substrate complexes are compared with the affinity of the phosphopyridoxal form of the enzyme for the amino acids, as estimated by competitive inhibition studies. Although ar-aminobutyrate was found to bind effectively to the enzymic active site, it reacted at only about 2% of the rate at which glutamate reacted. Because these two substrates differ only with respect to the y-carboxyl group of glutamate, this group must contribute to the reactivity of the enzyme-glutamate complex.

Alanine aminotransferase is highly specific for alanine and glutamate. In this paper we show that a formate-alanine amino- transferase-alanine (ternary) complex is formed which is more reactive than the binary complexes of the enzyme with either glutamate or alanine. Data are presented which suggest that formate binds to the enzymic site that normally influences the reactivity of the alanine aminotransferase-glutamate complex by interacting with the y-carboxyl group of glutamate.

101

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102 Alanine Aminotransjerase. II Vol. 242, No. 1

METHODS for which transamination was detected (Table I) were tested at

Assay Methods-Several assay procedures were used as indi- two different enzyme concentrations. Results showed that the

cated below. The extent of transamination in both directions amounts of alanine or glutamate formed were proportional to the

(alanine + cr-ketoglutarate + pyruvate + glutamate, and the enzyme concentration. None of the results can be attributed

reverse reaction from glutamate and pyruvate) can be determined to minor impurities in the substrates, for in all cases, excepting

by measuring the pyruvate concentration after a IO-min incuba- only ornithine (lOyO), the reactions were permitted to proceed

tion period with the enzyme by the salicylaldehyde assay method more than 20% toward the estimated equilibrium.

previously described (8). Amino transfer from alanine to o(- It was of particular interest to show transamination from

ketoglutarate (the forward reaction) was also measured by glycine by the alanine aminotransferase because of the expected

coupling the formation of pyruvate with lactate dehydrogenase- stimulatory effect not only of formate but also of acetate upon

catalyzed NADH oxidation. Similarly, the reverse reaction this reaction. For this purpose, a l-ml volume containing 50

was followed by coupling cY-ketoglutarate formation with gluta- mM highly purified glycine and 100 mM phosphate buffer, pH 6.9,

mate dehydrogenase-catalyzed NADH oxidation (8). Trans- was incubated with about 4 mg of the phosphopyridoxal form

amination of ol-aminobutyrate (Fig. 4) was followed by reduction of the enzyme. The absorbance at 425 rnp was 0.6. Trans-

of the ol-ketobutyrate formed in the reaction with NADH and amination to the phosphopyridoxal form of the enzyme may be

an excess of lactate dehydrogenase, purified 30.fold from extracts measured as a decrease in the absorbance at 425 rnp, since the

of whale muscle. The initial rates of transamination were de- phosphopyridoxamine form does not absorb at this wave length.

termined by observing the decrease in the absorbance at 340 rnp In the presence of glycine, after 24 hours at room temperature,

with a Gilford recording spectrophotometer. All assays were no decrease in the absorbance at 425 rnp was detected. Glycine

carried out at 37” in 0.1 M Tris buffer (pH 8.1 at 25”, which is transamination was, therefore, catalyzed by the alanine amino-

pH 7.8 at 37”). Specific activity was as previously defined (8). transferase at a rate more than 10 million times slower than was

Substrate Spec$city-Amino acid pairs were separated by that of alanine.

ascending paper chromatography. N-Butyl alcohol-pyridine- Column 2 of Table II summarizes the capacity of substrate

water (1 :I :I) was used for all separations except for the analogues to inhibit the transfer of the amino group of alanine

following. Aspartate was separated from glutamate in pyridine- to a-ketoglutarate. Inhibitions by aspartate, a-aminobutyrate,

acetic acid-water (10:7:3), ornithine and norleucine were norleucine, ac-aminoadipate, and glycine were not reduced by

separated from glutamate with N-butyl alcohol-acetic acid-water increasing the cr-ketoglutarate concentration lo-fold, but were

(12:3:5), and asparagine was separated from glutamate with reduced by increasing the concentration of alanine. Thus, the

phenol-water (5:l). The amounts of alanine or glutamate substrate analogues must inhibit by binding to the active site

produced were determined by the quantitative ninhydrin pro- of the phosphopyridoxal form of the enzyme.

cedure of Kay, Harris, and Entenman (9), except that neutral The capacity of the amino acids to inhibit glutamate trans-

(NaOH omitted) 0.5% ninhydrin spray was used (10). amination is tabulated in Column 3. It can be seen that those

Exchange Transamination-The rates of radioactive exchange analogues which inhibit alanine transamination most effectively

between analogous amino and keto acids were measured as are also the most effective inhibitors of glutamate transamination.

previously described (8). Fig. 1 shows that, as expected, a-aminoadipate inhibits trans-

Deutera’um Incorporation-The rates of deuterium incorpora- amination from alanine to an excess of a-ketoglutarate in a

tion into the (Y positions of the amino acids were determined by strictly competitive fashion.1 The cr-aminoadipate-enzyme

integrating the a-proton nuclear magnetic resonance peaks as dissociation constant is 0.09 M. An apparent Michaelis con-

functions of time with a Varian A-60 nuclear magnetic resonance stant for alanine of 25 mM, estimated from the intercepts on the

spectrometer. The temperature was maintained at 28” with ordinate, is in agreement with the value determined directly (8).

the Varian V-6057 variable temperature system. Assuming that the substrate homologues which reacted slowly

Spectra-A Cary model 15 spectrophotometer was used for with the enzyme inhibit alanine transamination competitively,

recording spectra. as did a-aminoadipate, one can show that

MATERIALS ‘WI (100 - %IJ Kz %I, 000 - %Id = K,

Purification procedures for alanine aminotransferase (specific activity, 340) (8), n-alanine aminotransferase (n-alanine:2- oxoglutarate aminotransferase, EC 2.6.1.10) from Bacillus

where %I is the percentage of inhibition, K is the dissociation

subtilis (specific activity, 60) (II), and aspartate aminotrans- constant of the enzyme-inhibitor complex (or its K,), and the

ferase (L-aspartate:2-oxoglutarate aminotransferase, EC subscripts denote two different competitive inhibitors. The

2.6.1.1) (12) have been described. Crystalline malate de- percentage of inhibition, %I, is therefore a measure of the

hydrogenase was a gift of H. A. Barker. o-Methylisourea binding constants.

hydrochloride was synthesized according to the procedure of Kurzer and Lawson (13), recrystallized from hot methanol, and

1 In reactions involving two substrates, “competitive” inhibi- tors may influence both the apparent Michaelis constant, K,,

neutralized immediately before use. Other materials were as and the apparent maximum velocity, V,,,, for one substrate

previously described (8). when the concentration of the other is kept, constant. However, if the concentration of the other substrate is chosen such that, even in the presence of the inhibitor, there is sufficient to saturate

RESULTS the enzyme, strictly competitive inhibition may be shown for the substrate whose concentration is varied.

Substrate Spec$city and Binding-The substrate analogues Only the parameter K,

is then affected.

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Issue of January 10, 1967 M. H. Saier, Jr., and W. T. Jenkins 103

With the use of this equation, along with the dissociation con- stant determined for the cY-aminoadipate-phosphopyridoxal enzyme complex and the data in Column 2 of Table II, the con- stants given in Column 4 were estimated. The dissociation constant, estimated by this method, for the enzyme-glutamate

oi t - 10

I I 0 .05

L- a-Amlnoadipate CM)

FIG. 1. Inhibition of alanine transamination by L-a-amino- adipate. Present in the l-ml reaction mixture were 10 rmoles of a-ketoglutarate and 100 pmoles of Tris-HCl, pH 8.1. The concentration of L-a-aminoadipate was varied in the presence of three L-alanine concentrations. The graph is in the form of a “Dixon plot” for competitive inhibitors (l/v against [I]). The negative value of the dissociation constant for the enzyme-a- aminoadipate complex is found on the abscissa below the point of intersection.

TABLE I

Substrate speci$city of pig heart alanine aminotransferase

From 1 to 50 ~1 of an enzyme solution having a specific activity of 340 were incubat(ed at 37” in a 0.5-ml solution containing 0.04 M keto acid, 0.04 M amino acid (unless racemic, in which case the total amino acid concentration was 0.08 M), and 0.10 M Tris-HCI buffer, pH 8.1. After incubation periods of from 5 min to 13 hours, 10 ~1 of the reaction mixtures were spotted onto Whatman No. 1 filter paper for chromatographic separation and subsequent calorimetric determination of the amino acids.

Amino acid

L-Alanine ............................ L-a-Aminobutyrate .................. L-Norvaline ......................... L-Norleucine. ........................ L-Ornithine .......................... L-Aspartate, ......................... L-Glutamate ......................... DL-&Hydroxyglutamate .............. Le-Aminoadipate. ................... Glycine, @-alanine, n-alanine, L-serine,

DL-o-methylserine, L-leucine ........ L-Asparagine, L-glutamine, DL-O-

phosphoserine, L-kynurenine, D-glU- tamate, DLiu-methylglutamate, y’ aminobutyrate .....................

-

I a

Activity

<eaction with Reaction with -ketoglutaratt pyruvate

% 100

1.4

0.08 0.04 0.13

<0.05

%

1.5 0.2

0.02 0.16

loo 0.5 0.36

<0.05

complex is in agreement with the value estimated spectro-

photometrically (8). Alanine Aminotransjerase-Carboxylate Anion Interactions-

Fig. 2 shows the stimulation of alanine transamination by formate, contrasted with the inhibition by acetate, propionate, and butyrate. High concentrations of butyrate appear to inhibit transamination completely, but complete inhibition was not seen with either acetate or propionate. A plot of l/velocity against the concentration of butyrate gave a straight line, as expected for competitive inhibitors.’

TABLE II

Inhibition of alanine and glutamate transamination by substrate analogues

Amino transfer from alanine to a-ketoglutarate is designated the forward reaction; that from glutamate to pyruvate, the re- verse reaction. Present in the reaction mixtures were 50 mu substrate L-amino acid, 5 mM substrate keto acid, 50 mu substrate analogue (unless racemic, in which case its total concentration was 100 mu), and 100 mM Tris-HCI, pH 8.1. When the rate of keto acid formation was determined by a coupled assay procedure, 0.1 mg of NADH per ml and a large excess of the coupling enzyme were also present. Incubations ‘were at 37”. Percentage inhibi- tions of the forward reaction were determined with the use of the salicylaldehyde assay and the lactate dehydrogenase-coupled assay. Values obtained by these two methods were within 5%, and were averaged to give the reported values. Percentage inhib- itions of the reverse reaction were determined with the use of the glutamate dehydrogenase-coupled assay and, also, the salicylalde- hyde assay. Values obtained by these methods, which usually differed by less than lo%, were averaged to give the reported values. Dissociation constants were calculated as described in the text. An “S” after the name of an amino acid indicates its susceptibility to alanine aminotransferase-catalyzed transamina- tion -

I 1. Substrate analogue

2. Forward reaction

Glycine ..................... L-Alanine, S. ............... L-a-Aminobutyrate, S ....... L-Norvaline, S .............. L-Norleucine, S ............. L-Ornithine, S .............. L-Leucine. .................. L-Serine ....................

%

7

28 34 53 27 20 17

L-Aspartate, S .............. L-Asparagine .............. L-Glutamate, S ............ L-Glutamine. .............. nL-a-Methylglutamate ..... DL-p-Hydroxyglutamate, S r-Aminobutyrate .......... Glutarate .................. L-a-Aminoadipate, S. ......

6 19 60

8 35 30

3 3

28

D-Alanine. ................. 3 n-Glutamate ............... 0

Inhibition -

0 Michaelis constant (8).

3. Reverse reaction

%

10 45 29 32 50

7

40

3

17

5 0

-

4. Calculated dissociation

constant

m‘w

500

285 90 70 30

550

25

90

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We have shown that at pH 6.9 an “abortive” pyruvate-phos- The inhibitory effect of formate at high formate concentrations phopyridoxal enzyme complex can form, but that under condi- may be due to interactions of formate with the enzyme at sites tions similar to those used in Fig. 2, cr-ketoglutarate does not other than the one responsible for the stimulation or to inhibition form such a complex (8). The stimulatory effect of formate of the half-reaction involving cY-ketoglutarate. cannot, therefore, result from competition with cr-ketoglutarate If stimulation of alanine transamination was a result of the

for the inhibitor-binding site. binding of formate to the enzymic y-carboxyl-binding site, for- The Michaelis constant for alanine was shown to be 28 mM, mate should inhibit transamination of glutamate, and this was

and that for ol-ketoglutarate was 0.4 mu under the conditions observed (Fig. 3A). Unfortunately, this result cannot be used in Fig. 2 (8). The enzyme was, therefore, essentially satu- directly correlated with the activation of alanine transamination rated with substrates, and formate stimulation must have re- because the assay conditions required lower substrate concen- sulted from a change in the maximum velocity of transamina- trations, and with lower substrate concentrations the stimula- tion. tory effect of formate on alanine transamination was not ob-

served owing to counteracting inhibitory effects (Fig. 3B).

0-l With the use of the glutamate dehydrogenase-coupled assay procedure at pH 6.9 (0.1 M phosphate), in which glutamate IL”

-4 dehydrogenase readily reduced a-ketoglutarate but not pyruvate, inhibitions by formate, acetate, and propionate were all found to be mixed (uncompetitive).

1 Although their binding constants are comparable, cy-amino-

butyrate reacts at only about 2y0 of the rate at which alanine reacts. The greater assay sensitivity obtained by coupling cr-aminobutyrate transamination with NADH oxidation per-

0’ I I I I / I I I .I .2 .3 .4 5 .6 7 .0 ++

Carboxylate Anion (M)

FIG. 2. The effect of short chain monocarboxylate anions upon the rate of alanine transamination. For the study of formate action, the I-ml reaction mixtures contained 0.4 M alanine, 0.04 M

or-ketoglutarate, and 0.1 M Tris-HCl buffer, pH 8.1. One-half as much substrate was employed for the investigations involving other carboxylate anions. The enzyme was added after the temperature reached 37”. After a 10.min incubation period, the reaction was terminated by the addition of alkali and the total pyruvate was determined. The experimental points for the formate curve are the averages of triplicate determinations.

100

80

$ 5 60 4 8

40

contained 0.1 M Tris-HCl buffer, pH 8.1,40 mM L-a-aminobutyrate, 4 mM cu-ketoglutarate, 0.3 mg of NADH, and an excess of lactate dehydrogenase. The reaction was initiated by addition of transaminase.

B 20 -1

I , I I 0.1 0.2 0.3 0.4 The 0.3

MONOCARBOXYLATE ANION (M) FORMATE (M)

FIG. 3. A, the effect of short chain monocarboxylate anions I upon the rate of glutamate transamination. Initial glutamate

I I I I 0 2 4 6 0 2 4 6 8 10

and pyruvate concentrations were 0.1 M and 0.01 M, respectively. B, the effect of formate upon the rate of alanine transamination at different substrate concentrations. The amino acid to keto

[ I/ 1-t [FORMATE]

acid concentration ratio was always 10, with the alanine concen- FIG. 5. A, a plot of the relative velocity, v/ve, against [v/v0 - l]/ trations as indicated in the figure. Excepting substrate concen- [formate] for the formate-induced rate increase in Fig. 1. B, trations, both the forward and reverse reactions were conducted a plot of the formate inhibition of cy-aminobutyrate transamina- under the conditions described in the legend to Fig. 2. The tion in a form comparable to that in A. The data were taken salicylaldehyde assay was used for pyruvate determination. from Fig. 4.

I I I

00 CB

01 .08 .I6 .32

Formate (M)

104 Alanine Aminotransferase. II Vol. 242, No. 1

FIG. 4. Inhibition of amino transfer from L-a-aminobutyrate to a-ketoglutarate by formate. The reaction mixtures (2.5 ml)

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Issue of January 10, 1967 M. H. S&r, Jr., and W. T. Jenkins 105

1.00 0.80

0.60

0.40

A;- A; -

A, 0.20

0.10

0.08

0.06 -K&o Glutarate*z Glutamate

FIG. 6. The effect of formate upon the rates of amino transfer from the amino acid substrates to the analogous radioactive keto acids. A, glutamate + a-ketoglutarate; B, alanine + pyruvate. Reactions were carried out at 37” in 0.5-ml volumes containing 0.3 M Tris-HCl buffer (pH 8.1), 0.6 M amino acid, and 0.06 M keto acid (approximately 15 PC per mmole). 0, experimental points obtained in the absence of formate; 0, those obtained with 0.1 M formate present. The velocities of exchange transamination are proportional to the slopes.

mitted the effect of formate on transamination of this substrate analogue to be studied. The inhibition which was observed (Fig. 4) must be due only to an effect on the aminobutyrate half- reaction, since the half-reaction with ketoglutarate cannot be rate limiting. The shape of the curve obtained (Fig. 4) re- sembles those for acetate and propionate inhibition of alanine transamination (Fig. 2).

In the presence of a constant substrate concentration, formate, F, has an apparent dissociation constant K’f = [E’][Fj/[E’F] where [E’] = [E] + [ES] and [E’F] = [EF] + [.&SF]. Assum- ing further that the rate with an excess of formate, vf , differs from that in the absence of formate, 00, one may derive an ex- pression relating the observed rate, v, to the formate concentra- tion, for

u = Wluo + [E’FIQ = vo + v,wm; WI + [E’FI 1 + WI/K;

Therefore,

Vf 21 -=- + (v/v0 - 1)K; vo uo VI

or

V (1 - vlvo)K/ -= VO [F] + :

Fig. 5A shows that if v/v0 is plotted against (v/v0 - l)/(for- mate) for the data obtained with formate in Fig. 2, a straight line is obtained in the region of low formate concentration where inhibitory effects are slight. The value of K’f, which may be derived from the slope, is 0.04 M, and the ratio, V~:VO, is 1.25. The theoretical maximum increase in the rate caused by the addition of formate under these conditions is, therefore, 25% of vo.

Inhibition data may be similarly treated. The data for the formate inhibition of aminobutyrate transamination (Fig. 4) yielded the straight line shown in Fig. 5B. In this case, the

\

0 8 16 24

Minutes

corresponding values were K’f = 0.05 M and of/v0 = 0.40. Thus, within the limits of experimental error, the apparent affinity of the enzyme for formate is the same for both activation of alanine transamination and inhibition of aminobutyrate transamination.

If monocarboxylate anions of more than 1 carbon atom inhibit alanine transamination by binding to the same site to which formate binds, increasing the formate concentrateion, in the pres- ence of a constant concentration of one of these anions, should result in their displacement by the formate, and hence in acceler- ation of the reaction rate. This was observed. In the presence of 0.2 M acetate, propionate, or butyrate, the formate concentra- tion which caused the maximal rate of pyruvate formation was found to be about 0.4 M. It will be recalled that when formate was the only monocarboxylate anion present in the reaction mixture, much less formate (0.1 M) accelerated maximally (Fig. 2).

In order to pursue the differential effect of formate upon both alanine and glutamate transamination under comparable condi- tions, “exchange transaminations” between alanine and pyru- vate and between glutamate and ketoglutarate were studied (Fig. 6). In both cases, the concentration of the amino acid, [A], was 0.6 M, and that of the analogous keto acid, [K], was 0.06 M. The lower concentration of keto acid minimizes the forma- tion of the abortive complex between pyruvate and the phos- phopyridoxal enzyme. It also improves the accuracy with which the extent of the reaction may be measured.

The basic equation for radioactive exchange transamination is d[A*]/dt = u([K*]/[K] - [A*]/[A]), where v is the velocity of the exchange reaction and [A*]/[A] and [K*]/[K] are the specific activities of the amino acid and keto acid, respectively. Upon integration, this equation gives the linear equation

0 20 40 60

MINUTES

FIG. 7. The effect of formate on the rates of deuterium incor- poration into the substrate amino acid a-carbon atoms as deter- mined by integration of the a-proton nuclear magnetic resonance peaks. Acids were neutralized to pD 8.5 with sodium carbonate. Reactions were carried out at 23” in 0.5-ml volumes containing 0.6 M amino acid and 0.06 M analogous keto acid. From 5 to 10 integrations were taken at 2-min intervals, and the average values were obtained. These experimental values lay close to smooth curves when plotted with respect to time. The points shown were taken from these curves. Integrations were corrected for the small cr-hydrogen resonance peak which remained after equi- libria were attained. The slopes are proportional to the velocities of a-hydrogen exchange in DzO.

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106 Alanine Aminotransjerase. II Vol. 242, No. 1

-In [A,*1 - [A,*1

[&*I > = (1 + R)vtlM

where R is the amino acid to keto acid concentration ratio, [A,*] is the concentration of radioactive amino acid after equilibrium

.I0 .I5 .20 .25 .30 35

Carboxylate Anion (M)

FIG. 8. The effect of monocarboxylate anions on the rate of transamination from n-alanine to a-ketoglutarate. Curves A and B represent data obtained when the substrate concentrations were 0.4 M n-alanine and 0.04 M ketoglutarate. Curves C and D, substrate concentrations were 0.2 M n-alanine and 0.02 M keto- glutarate. Curves A and C illustrate the effect of acetate; B and D show the effect of formate. Other reaction conditions were as for Fig. 1.

10

a

I I I ADDITION

1

/

None

Butyrate

/ /v Propionatl ei

Formate 6.8

I/Vc +

1.0

1.4

2.3

l/[L-alanmel

FIG. 9. Stimulation of aspartate aminotransferase-catalyzed alanine transamination by 0.4 M carboxylate anions. Present in the 2.0.ml reaction mixtures were 200 pmoles of Tris-HCl (pH 8.1), 0.2 mg of NADH, 20 pmoles of or-ketoglutarate, excess lactate dehydrogenase, and various amounts of r,-alanine. Reactions were initiated by addition of aspartate aminotransferase after temperature equilibration at 37”.

is reached, and [A,*] is the concentration of radioactive amino acid at time t. A graph of the left hand term with respect to time gives the velocity, since R and [A] are known.

It can be seen from Fig. 6B that the alanine-pyruvate exchange reaction was accelerated by the presence of 0.1 M formate to the extent of 20%. Fig. 6A illustrates that the glutamate-keto- glutarate exchange reaction was inhibited by 0.1 M formate. In this case, the rate dropped to 91%. Quantitatively similar results were obtained in several different experiments, although slight differences were noted in the degree of activation and in- hibition. These experiments showed that, in the absence of formate, exchange transamination between alanine and pyruvate proceeded somewhat more rapidly than that between glutamate and Lu-ketoglutarate.

In an attempt to define more clearly the specific step in the amino transfer reaction affected by formate, the rates of exchange of the a-hydrogen atoms of the amino acid substrates with the medium were studied. Because the hydrogen atoms of pyruvate exchange with the medium, incorporation of tritium from the medium into the cy position of the substrate amino acids could not be followed. However, the removal of the or-hydrogen atoms in deuterium oxide could be studied by following the decrease in the nuclear magnetic resonance’peaks ascribed to the a-hydrogen atoms.

In this case the basic equation for the replacement of the a-hydrogen atom, [a-H], with deuterium is d[cr-H]/dt = -+[a-H]/ [A] where v is the rate of a-hydrogen exchange, and [A] is the amino acid concentration. Upon integration the following equation is obtained.

The term in parentheses is the ratio of the a-proton “concentra- tion” at time t to that at zero time. Plotting the left hand term against time should give a straight line whose slope is propor- tional to v. Fig. 7 reveals that 0.1 M formate accelerated the rate of exchange of the alanine a-hydrogen atom by 15%, while that of glutamate was inhibited by 13%. Furthermore, in the absence of formate the rate of exchange of the alanine cr-hydro- gen atom proceeded somewhat more rapidly than was observed for the glutamate a-hydrogen atom. These relative rates agree, within the limits of experimental error, with those determined for radioactive exchange transamination. The velocities of radioactive exchange, calculated from the data in Fig. 6, were of the same order of magnitude as the velocities of a-hydrogen atom exchange for the same amount of enzyme. These data are consistent with the supposition that the slow step in both half-reactions involves breakage of the carbon-a-hydrogen bond.

With much higher concentrations of enzyme, the enzyme- substrate complexes may be observed spectroscopically. No effect of formate at concentrations of 0.06 and 0.12 M was noted in the presence of mixtures of alanine (0.5 M) and pyruvate (0.05 M) at pH 8.1 (Tris-HCl). Since the spectral maxima ob- served under such conditions are thought to represent several enzyme-substrate complexes in equilibrium (14), this result indicates that formate binding does not appreciably alter the equilibria between those complexes which are present in suffi- cient concentrations to be observed.

Formate slightly decreased the heights of the spectral peaks observed in the presence of glutamate (0.5 M) and ar-ketoglutarate

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Issue of January 10, 1967 M. H. Xaier, Jr., and W. T. Jenkins 107

(0.05 M). A reduction in the equilibrium concentrations of the enzyme-substrate complexes due to competition by formate for a substrate-binding site would account for this result.

Investigations with Other Enzymes-In order to investigate the possibility that formate stimulation was a general feature, rather than a unique property of soluble alanine aminotransferase from pig heart, other aminotransferases were studied. As shown in Fig. 8, both formate and acetate stimulated n-alanine aminotransferase-catalyzed amino transfer from n-alanine to ry-ketoglutarate with high substrate concentrations, and in- hibited with lower levels.

Fig. 9 shows that all of the monocarboxylate anions tested stimulated aspartate aminotransferase-catalyzed alanine trans- amination by increasing V,,,,/K,, i.e. decreasing the slope of a l/v against l/[S] plot. Whether or not this is due solely to an effect upon the maximum reaction velocity, I’,,,, as was found to be the case for alanine aminotransferase-catalyzed alanine transamination, cannot be established because the Km of the alanine-aspartate aminotransferase complex is very large and cannot be determined accurately. The relative reaction veloci- ties in the absence and in the presence of the various anions (0.4 M), calculated from the slopes, are included in the figure. Formate is seen to accelerate most effectively; butyrate, least effectively.

Aspartate aminotransferase-catalyzed amino transfer from glutamate to pyruvate was not stimulated by formate, even though the conversion of the phosphopyridoxamine form of the enzyme and pyruvate to the phosphopyridoxal form of the en- zyme and alanine had to be rate-limiting. Nor was the rate of amino transfer from aspartate to ol-ketoglutarate increased by adding formate.

In the presence of 10 mM a-ketoglutarate and 100 mM amino acid substrate under the conditions described in the legend to Fig. 9, aspartate aminotransferase catalyzed aspartate trans- amination about 50,000 times more rapidly than alanine trans- amination. In these investigations, the rates of amino transfer from aspartate to ar-ketoglutarate were determined by coupling the formation of oxalacetate with malate dehydrogenase-cata- lyzed NADH oxidation.

Protein Derivatization-An attempt was made to inhibit the glutamate half-reaction selectively by modification of lysine e-amino groups on the assumption that such a group was involved in y-carboxyl binding. o-Methylisourea has been shown to be highly specific for the e-amino function of lysine (15, 16). There- fore, the effect of guanidinating exposed lysine residues in alanine aminotransferase on the rates of alanine and glutamate trans- amination was studied. The formation of pyruvate or cr-keto- glutarate was coupled with NADH oxidation with lactate dehy- drogenase or glutamate dehydrogenase, respectively.

Guanidination was carried out at pH 9.4 and 4” with 0.5 M

o-methylisourea, 2 mM dithiothreitol, a trace of EDTA, and 0.5 mg of protein per ml, present in the reaction mixture. The enzyme, which was stable under these conditions in the absence of the reagent, was totally in the phosphopyridoxal form. Ali-

quots were periodically removed, brought to pH 6.5 with phos- phate buffer (which terminated guanidination and inactivation), and assayed by the two procedures. The slow rates of loss of alanine- and glutamate-transaminating activities were the same (30% inactivation after 24 hours). No information about the y-carboxyl-binding site was therefore obtained from this ap- proach.

DISCUSSION

All of the transaminations reported in Table I are believed to be due to catalysis by alanine aminotransferase. Results were shown not to be due to trace impurities in the substrates, and the possibility that aspartate aminotransferase, leucine aminotrans- ferase, and other aminotransferases known to be present in pig heart (17) contaminated the enzyme preparation is eliminated by the data.

It should be noted that higher and lower homologues of L-

glutamate and n-alanine were transaminated at much reduced rates; the more the chain length differed from those of the natural substrates, the more the rate decreased. It is interesting that, for homologues of alanine, the affinity of the enzyme for the amino acid increased with increasing chain length while reactivity decreased. Because the specificity studies were conducted under conditions insufficient to saturate the enzyme with the amino acids, the effect of chain length would be expected to be more pronounced on the maximum transamination velocities than on observed velocities.

It is possible that there are separate enzymic binding sites for the a-carbon substrates (alanine and pyruvate) and for the 5- carbon substrates (glutamate and a-ketoglutarate). Alterna- tively, all four substrates may bind to a set of amino acid resi- dues which constitute a single binding site, with the amino or keto acid side chains in the same position relative to enzymic amino acid residues and to the remainder of the substrate molecule.

Those substrate analogues which most effectively inhibited alanine transamination inhibited glutamate transamination comparably (Table II). Furthermore, Fig. 1 shows that L-W

aminoadipate, the first higher homologue of glutamate, inhibited alanine transamination competitively, and we have previously shown that pyruvate is a competitive inhibitor of both alanine and glutamate transamination (8). These data support the hypothesis that independent binding sites for alanine and glu- tamate do not exist on the phosphopyridoxal form of the enzyme.

Stimulation of alanine aminotransferase-catalyzed transamina- tion was observed only with formate as carboxylate anion and alanine as amino donor, and cannot, therefore, be explained by nonspecific interactions with the enzyme protein. We conclude that formate affects the rate by binding to a site which determines the specificity of the enzyme.

The finding that acetate and propionate inhibit alanine trans- amination, but not competitively, may be correlated with the comparable inhibition by formate of a-aminobutyrate trans- amination. Thus, the carboxyl groups of the monocarboxylate anions must bind to an enzymic site in a manner that leads to interaction between the substrate and monocarboxylate side chains. The y-carboxyl binding site is a likely candidate for this site. The different effects of formate on the two exchange transamination reactions substantiate this conclusion.

One might expect that formate and a-aminobutyrate would fit into the same space as glutamate, but examination of space- filling molecular models (Fig. 10) reveals that steric hindrance between the hydrogen atom of formate and the methyl group of aminobutyrate prevents this. Overlap similarly occurs with acetate and alanine. Both the stimulation of alanine transamina- tion and the inhibition of a-aminobutyrate and alanine trans- aminations are, therefore, understandable in terms of the pro- posed stereochemistry of the enzyme-substrate intermediates. (This explanation tacitly assumes that alanine and glutamate bind to the same enzymic site before reacting, an assumption

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Alanine Aminotransferase. II Vol. 242, No. 1

FIG. 10. Space-filling molecular models which illustrate the steric interactions which may exist in the formate-alanine amino- transferase-substrate ternary complexes. Left, alanine and formate; center, glutamate; right, aminobutyrate and formate. In all cases the or-amino and cr-carboxyl groups were omitted for simplicity. The side chains are in the staggered configuration.

discussed above.) A comparable result was obtained by Ina- gami and Murachi (5). They showed that while propylam- monium ion stimulated tryptic hydrolysis of acetylglycine ethyl ester, the butylammonium ion inhibited. Lysine, present in the natural substrate, has a side chain the length of the butylam- monium ion.

In contrast, both n-alanine aminotransferase- and aspartate aminotransferase-catalyzed alanine transaminations were stimu- lated by acetate as well as formate. If steric hindrance was re- sponsible for the results observed with alanine aminotransferase, less rigid orientation requirements for the former two enzymes or separate binding sites for alanine and glutamate must be as- sumed. If the former is correct, the carboxyl group of the mono- carboxylates might be bound in such a way that the aliphatic chain could be displaced away from the site at which alanine is bound without loss of the stimulatory effect. The relatively wide specificity requirements of n-alanine aminotransferase (ll), and the observation that even propionate and butyrate stimulated the aspartate aminotransferase-catalyzed reaction, are consistent with either explanation.

Comparison of the transamination rates of cu-aminobutyrate and of glutamate showed that y-carboxyl binding had to be re- sponsible, in part, for the reactivity of the glutamate-enzyme complex, but the alanine-enzyme complex was found to be slightly more reactive. Two explanations of formate stimulation initially appeared to be most reasonable. First, the rate-limiting step in the transamination of alanine was not necessarily that which limited the rate of glutamate transamination. If they were to differ, the binding of formate to the enzyme-alanine complex might be expected to increase the rate of the slow step of alanine transamination toward the rate of the comparable step with the glutamate complex. However, the nuclear magnetic resonance studies of the a-hydrogen exchange reaction indicated that re- moval of the a-hydrogen atom’is the rate-limiting step for trans- amination of both substrates, and, therefore, that this explana- tion is not the correct one. The second possibility is that, while y-carboxyl binding increases the reactivity of the enzyme-sub- strate complex, the /3-methylene group of glutamate has an

“anticatalytic” effect on the rate. That is, the presence of this group decreases the reactivity of the enzyme-substrate complex. This hypothesis also explains the slow rates at which higher homo- logues of alanine are transaminated.

The stimulations described in this and other papers (l-5) can be explained by assuming that introduction of the stimulator into a specific enzymic site increases reactivity by bringing about ap- propriate orientation of protein amino acid residues involved in catalysis. Thus, the stimulations as well as the inhibitions of the transaminase-catalyzed reactions caused by carboxylate anions appear reasonable in terms of the “induced fit” hypothesis origi- nally proposed by Koshland to explain enzyme substrate speci- ficity data (18-20; also see Reference 21). Other experimental approaches which have shed light on the problem of enzyme sub- strate specificity (see, for example, References 22 and 23) further support the conclusion that our results are reasonably inter- preted in terms of this hypothesis.

Aci&owZedgments-We wish to thank Dr. D. E. Koshland and Dr. C. E. Ballou for helpful discussions and for reading the manuscript.

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Milton H. Saier, Jr. and W. Terry JenkinsAlanine Aminotransferase: II. THE BASIS FOR SUBSTRATE SPECIFICITY

1967, 242:101-108.J. Biol. Chem. 

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