alanine racemase of pseudomonas: observations on subtrate and inhibitor specificity

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 165, 126-132 (1974)

Alanine Racemase of Pseudomonas

Observations on Subtrate and Inhibitor Specificity’

ELIJAH ADAMS,2 KANAI L. MUKHERJEE,3 AND HARMON C. DUNATHAN

Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201, and Department of Chemistry, Haverford College, Haverford, Pennsylvania, 19041

Received April 15, 1974

Systematic studies with purified alanine racemase and a number of substrate analogs permit the generalization that effective competitive inhibition is limited to 2- and 3-carbon compounds. A free o-amino group was not necessary for relatively tight binding; compounds lacking an amino group, or with an a-amino group acylated even by a bulky substituent, were bound as tightly as alanine. Substitution at the o-carbon of alanine (i.e., replacement of the cu-H) eliminated binding, while substitution at the p-carbon generally reduced binding. Of several inhibitory compounds tested for substrate activity by H exchange with “H,O, only glycine appeared active. Covalent binding to the enzyme by halo analogs was not demonstrated.

The amino acid racemases, at present known only as enzymes of bacteria, have in general not been well-characterized with respect to kinetic and specificity features (1). In particular, alanine racemase, proba- bly the most widely distributed enzyme in the group, has been described as a homogeneous protein only in recent, brief reports (2, 3), and only fragmentary data are available concerning substrates and inhibitors (4, 5). We report here a systematic study of structurally related compounds as inhibitors of alanine racemase of Pseudomonas. Certain of these compounds have also been tested as substrates by hydrogen exchange. In addition, we present data concerning the stability of the enzyme in fractions of varying purity.

MATERIALS AND METHODS

Source of Enzyme

The source, preparation, and purification of alanine racemase were as described earlier (3); the earlier

‘Supported by research grants from the U. S. Public Health Service to Elijah Adams (GM-11105) and to Harmon Dunathan (GM-20184).

?To whom correspondence should be addressed. 3 Present address: Essex Community College, Bal-

timore, MD 21237.

purification procedures were reproducible except for the present finding that, during the last two steps of purification (3), addition of EDTA (lOma M) to all solutions in contact with the enzyme appeared to favor obtaining homogeneous enzyme. Because of the instability of the final fraction of homogeneous enzyme obtained from Ecteola-cellulose, all of the kinetic data reported here were obtained with the prior fraction (DEAE-cellulose), judged to be about 25% pure as a protein (3).

Enzyme Assays

The assay for n-alanine formation from L-alanine was as described earlier (3), by the measurement of n-alanine formed in an incubation mixture with the racemase (Incubation I); a small aliquot of Incubation I was added to a mixture of n-amino acid oxidase, lactate dehydrogenase, and NADH (Incubation II), and the quantity of NADH oxidized was a measure of the o-alanine in that aliquot. The assay for L-alanine formation from n-alanine was essentially similar to that in the other direction except that for the measurement of L-alanine as a reaction product, Incuba- tion II contained glutamic-alanine transaminase, or- ketoglutarate, lactate dehydrogenase, and NADH, much as described by Lambert and Neuhaus (5). Suf- ficient enzyme to catalyze the limiting step in Incuba- tion II (either crystalline D-amin acid oxidase for the detection of n-alanine or the transaminase for the detection of L-alanine) was added to complete the measurement of either antipode of alanine, in the ali-

126

Copyright 0 1974 by Academic Press. Inc. All rights of reproduction in any form reserved.

BINDING SPECIFICITY OF ALANINE RACEMASE 127

quots used, in about 10 min at 40°C. Sources and quantities of enzymes were: n-amino acid oxidase (crystalline, hog kidney) from Sigma, 1.0 unit per assay mixture; glutamic-alanine transminase (pig heart) from Sigma, 10 units per assay mixture; lactate dehydrogenase (crystalline, rabbit muscle) from Sigma, 85 units per assay mixture. That only D- or L-ala-

nine (and not pyruvate) was formed in Incubation I was shown by the time course of the coupled reaction in Incubation II, indicating a relatively slow genera- tion of pyruvate. Had preformed pyruvate been present in the aliquot of Incubation I, the large excess of lactate dehydrogenase in Incubation II would have catalyzed an instantaneous oxidation of NADH: this was not observed. Protein was determined as before (3). One unit of enzyme is defined as the quantity re- quired for the formation of 1 pmole of product per minute under the assay conditions used.

Source of Compounds

All compounds tested were purchased from standard sources such as Sigma or Calbiochem, except for j3-chloro-L-alanine, e-aminocaproyl-L-alanine and L-

lysyl-L-alanine. p-Chloro-L-alanine. &Chloro-L-alanine was pre-

pared as originally described (6); because the amino acid hydrochloride has only low rotation (6), free /?-chloro-L-alanine was prepared from the hydrochloride as described in order to check optical purity by rotation (6). The specific rotation was found to be -15.6” (0.3 M in water; 25°C); specific rotation cited (6): - 15.5”.

r-Ami~caproyl-L-alanine. c-Aminocaproyl-L-alanine and L-lysyl-L-alanine’ were prepared by carbodiimide coupling as follows. N-Carbobenzoxy-e- aniinocaproyl-L-alanine methyl ester was prepared by mixing 5 mmoles of carbobenzoxy-r-aminocaproic acid (Sigma Chemical Co.) in 10 ml of CHCl, with 5 mmoles of L-alanine methyl ester hydrochloride (Sigma Chemical Co.), neutralized with an equivalent of triethylamine, in 10 ml of CHCI,. The combined solution was chilled in ice, 5 mmoles of dicyclohexyl- carbodiimide were added, and the mixture was kept at 5°C overnight. Crystals of dicyclohexyl urea were removed by filtration, and the filtrate was washed successively with 1 N HCl, 1 N NaHC03, and water. After drying over Na,SO,, the CHCl, solution was concentrated to an oil by flash evaporation, brought up in ethyl acetate, and filtered to remove a further crop of the urea. The filtrate was evaporated to an oil, triturated with n-heptane, and kept at 5°C. Crystals which formed were filtered and dried in vacuum. Yield, 1.6 g (94%), mp 66-69°C. We subsequently noted that this compound had been prepared earlier,

‘We thank Mr. C. L. Hsiao, a predoctoral student at the University of Maryland, for his participation in the preparation of these two dipeptides.

by way of the acyl chloride of carbobenzoxy-c- aminocaproic acid (7); reported mp, 67°C.

Carbobenzoxy-e-aminocaproyl-L-alanine was prepared by saponification of 1.23 g (3.5 mmoles) of the methyl ester in a mixture of 15 ml of methyl alcohol and 5 ml of 1,4-dioxane, to which 2 ml of 2 N NaOH were added. The solution was kept at 37°C for 2 hr, then filtered, treated with 3 vol of water, and acidified with 6 N HCl. The product crystallized immediately, and after 18 hr at 5°C was collected by filtration and dried. Yield 0.5 g (54%), mp 74°C. The carbobenzoxy dipeptide is also cited in (7), but no melting point is given. The free dipeptide was obtained by hydrogenolysis of the carbobenzoxy dipeptide above (230 mg) in 50 ml of methyl alcohol, containing 2 ml of acetic acid and 200 mg of Pd.charcoal catalyst (Matheson. Cole- man and Bell). The reaction was complete (cessation of CO, production) in 1 hr. The suspension was filtered, the filtrate was evaporated to an oil and treated with ether and heptane. The solid residue was dried to yield 80 mg (64%).

L-Lysyl-L-alanine. The dipeptide, L-lysyl-L-alanine, was prepared by essentially similar steps, first coupling dicarbobenzoxy-L-lysine (Sigma Chemical Co.) with L-alanine methyl ester hydrochloride and isolating the product, dicarbobenzoxy-L-lysyl-L-alanine methyl ester, essentially as above. The blocked dipeptide was saponified as above to obtain the dicarbobenzoxy dipeptide; this was then hy- drogenated to obtain the free dipeptide. From 5 mmoles of dicarbobenzoxy-L-lysine and 5 mmoles of L-alanine methyl ester, we obtained 1.8 g (75%) of the blocked dipeptide, mp 133-136°C. The carbobenzoxy dipeptide methyl ester (2.5 mmoles) yielded 1.12 g (95%) of dicarbobenzoxy-L-lysyl-L-alanine. mp 145-148°C. Hydrogenolysis of 200 mg of the above compound yielded 60 mg (67%) of the free peptide. The synthesis of dicarbobenzoxy-L-lysyl-L-alanine ethyl ester (mp 119OC) via the azide, saponification of this to yield dicarbobenzoxy-L-lysyl-L-alanine (mp 15O”C), and hydrogenolysis to the free dipeptide have been described previously (8).

a-H Exchange as a Test of Substrate Activity

Compounds tested as substrates were incubated in ‘H,O with large quantities of the racemase as outlined in detail in Table III. Incorporation of radioactivity into compounds tested was measured directly (after chromatographic isolation) in the case of glycine and alanine, since these appeared to incor- porate 3H. In the other compounds tested (Table III), residual radioactivity after at least 5 cycles of evaporation to dryness was taken as a measure of incorporation. Validation of the latter measurement was based on essentially similar results for glycine and alanine whether incorporation was measured by residual radioactivity after evaporation or by the more elaborate procedure of isolating these amino acids

128 ADAMS, MUKHERJEE AND DUNATHAN

after thin-layer chromatography. In addition, in all cases tested, 3 cycles of evaporation yielded residual radioactivity which did not change with further evaporation; 5 cycles of evaporation was therefore routinely employed. Although some of the compounds tested are volatile as the free acids, evaporation at the pH of the assay (7.8) should have insured their novol- atility as salts.

RESULTS

Stability of Purified Fractions

It was reported earlier that the final two purification fractions (Fractions 6 and 7 (3)) were unstable on storage at -15”C, with or without added nL-alanine, thiols, EDTA, or pyridoxal phosphate. A more sytematic test of stability indicated that Fraction 6 (DEAE-cellulose) was quite stable at -7O”C, with or without added EDTA or pyridoxal phosphate, but that Fraction 7 (Ecteola) was unstable at all temperatures and with all additions tested. Decay rates of both fractions appeared to be first order. Curiously, the storage temperature (either -10” or -70°C) did not affect the decay rate of the final purified fraction, nor did storage at -196°C pre- serve enzyme activity. Some of these findings are summarized in Table I.

TABLE I

STABILITY OF FINAL FRACTIONS OF ALANINE RACEMASEO

Fraction Temperature Half-life (” C) (days)

6 5 2.6 6 -10 4.0 6 -70 > 50b

7 -10 2.4 7 -70 2.4 7 - 196 not determinedc

“Fractions 6 and 7 (3) were assayed when fresh, and were then stored, without concentration, at the temperatures shown. Separate tubes were removed for assay at 3-5 time intervals for all entries except Fraction 7 at -196°C. The points fell on a first-order decay line. Various additions including pyridoxal phosphate (0.901 M), dithiothreitol (0.02 M), or 0.1 M NaCl (in order to bring Fraction 7 to the approximate NaCl concentration of Fraction 6) were without effect on the stability of either fraction.

* No activity lost at 45 days. =A11 activity lost at 45 days.

Kinetic Constants

Lineweaver-Burk plots for the reaction with L- or n-alanine as the initial substrate gave rather similar kinetic constants. In approximate agreement with an earlier determination for the homogeneous enzyme (3), fraction 6 yielded K, values for L-alanine (two determinations) of 0.020 and 0.033 M. The Km’s for n-alanine, not previously reported for this enzyme, were 0.016 and 0.014 M from comparable data. The respective ratios for VI/V, were approximately 1.8 in two determinations. The equilibrium constant, calculated from the appropriate Haldane expression, V,KDIVDKL, averaged 1.1. Preliminary ex- periments have shown a substantial CX-~H kinetic isotope effect on V in the L + D

direction but with no effect on K,.

Znhibition Studies

A number of compounds were screened for significant inhibitor activity by measur- ing enzyme activity with 0.1 M L-alanine as substrate in the presence of 0.05, 0.1, and 0.2 M concentrations of prospective inhibitor. In all cases, the compounds tested were neutralized with NaOH, where necessary, to pH 7-8, then added to the incubation mixture, buffered as usual. The pH of Incubation I was shown not to be altered by the addition of any compound tested. That inhibitors acted on the racemase rather than on the enzymes for n-alanine assay (Incubation II) was based on the nature of the assay. Incubation II measures the quantity of n-alanine formed, and no apparent inhibition of the rate of this reaction (D-alanine + pyruvate + lactate) was observed with the small aliquot (usually 0.01 ml) of Incubation I transferred to In- cubation II.

Both noninhibitory and inhibitory compounds are shown in Table II. Some of these, selected for special structural interest or because of relatively strong inhibition, were examined in greater detail: for these, values of Ki are shown. Whenever examined in this way, inhibition proved to be clearly competitive by the usual inverse plot criteria. Not shown in Table II is p- chloro-L-alanine, which appeared to react


TABLE II

INHIBITORS OF ALANINE RACEMASE’

Inhibitor

Acetate Glycine Propionate Acrylate

58 - 40 130 83 15 95 5

N-Acetyl-L-alanine L-Alanyl-L-alanine p-Alanyl-L-alanine c-Aminocaproyl-L-alanine L-Lysyl-L-alanine

91 57 43 90 72

14

-

38 46

2,3-nL-Diaminopropionate @-Alanine 3Xhloropropionate L-Cysteine Butyrate Valerate

Glycylglycine L-Lactate 2-Bromopropionate

0.2 0.2 0.2 0.1

0.2 0.1 0.2 0.2 0.2

0.2 0.2 0.2 0.2 0.2 0.2

0.2 0.2 0.2

0.2 0.2

0.01

48 37 73 48 36 69

- - - - -

58 -

57 -

99 25

L-Alanine methyl ester oL-Alaninol

I-Aminoethylphosphonate

50 44

45

- -

0.55 Not inhibitory at 0.2 M: L-a-aminobutyrate, 2-aminoethylphosphonate, a-aminoisobutyrate, L-cysteate, L-lysine, L-norvaline (0.1 M), L-praline, L-serine, taurine, L-tyrosine

Concen- Inhi- K, tration bition (mu)

(M) (%I

D L-Alanine was present at 0.1 M in each case. The uninhibited rate was about 25 units/ml enzyme (Frac- tion 6 (3)). Compounds tested are arranged in the Table in approximately the order in which they are considered in the Discussion.

in the assay by giving values higher than the uninhibited reaction, apparently as a re- sult of a reaction in Incubation II. This reaction, unexpected because of the optical specificity of D-amino acid oxidase, is under investigation.

In addition, several sulfhydryl reagents were tested as possible inhibitors. These included p-chloromercuribenzoate (0.9 mM inhibited completely; 0.01 InM inhibited 50%), N-ethylmaleimide (noninhibitory at 10m3 M), and sodium iodoacetate and sodium arsenite (.both noninhibitory at 10e2 M).

Reversibility of Inhibition

Certain of the inhibitors above were tested for time-dependent irreversibility of inhibition in order to detect possible formation of a covalent bond with the enzyme. Compounds so tested were acrylate, 1-aminoethylphosphonate, 2-bromopropionate, 3-chloropropionate, and propionate. Each inhibitor was incubated with the enzyme in the absence of alanine at a concentration which would inhibit the initial rate of 0.1 M L-alanine racemization approximately 50%. Incubation conditions otherwise were those of the Etandard assay. At intervals of 1, 2, and 4 hr, samples of the enzyme-inhibitor solution were assayed with 0.1 M L-alanine. Control loss of enzyme activity, attributable only to the incubation conditions, was estimated by parallel incubations without inhibitor: in 4 hr, enzyme inactivation averaged 25%. No loss of activity beyond this control percent occurred with any of the inhibitors tested,

Substrate Activity of Inhibitors

It was of interest to test certain of the inhibitory components as substrates. Since the standard assays were suitable only for the racemization of D- or L-alanine, a general assay based on the presumptive exchange of the a-hydrogen of a substrate was adopted. It should be noted that little information is available concerning the labilization of the a-hydrogen of an amino acid substrate as a concomitant of enzymatic racemization by the pyridoxal phosphate racemases5. Data previously reported, however, showed that exchange was far less than stoichiometric, compatible with substantial isotope discrimination against the replacement of an (Y-H with the tritium of water (11, 12). For this reason, tests of substrate activity by the method used could be only qualitative.

By the method detailed in Table III, substrate activity was examined for gly- tine, acrylate, 1-aminoethylphosphonate,

‘Extensive studies of hydrogen exchange have been reported both for praline racemase (9) and hydroxyproline-2-epimerase (IO); however, it is likely that the mechanism of these reactions differs from that of racemases for primary amino acids (1).


TABLE III

HYDR~CEN EXCHANGE AS A TEST OF SUBSTRATE

ACTIVITY”

Compound incubated

Alanine Glycine 1-Aminoethyl-

phosphonate’ 2-Bromopropi-

onate Propionate Acrylate N-Acetyl-L-

alanine &Chloro-L-

alanine P-Chloro-L-

alanined

Specific activity0

No Heated Active enzyme enzyme enzyme

dpm/pmole - 70 105

2 x 101 5 x 103 9 x 10’ 102 102 102

6 x lo3 6 x lo2 2 x 102

60 60 60

103 2 x 10s 2 x 103 2 x 103 2 x 103 103

3 x 105 2 x 10” 3 x 105

10’ 10’ 10’

a All compounds were incubated at a final concentration of 0.1 M (except as noted) in a final volume of 0.5 ml containing 0.02 M Tris-HCl (pH 7.8), 15 mCi of 3H,0 and 6 units of enzyme (Fraction 6). After 16-18 hr incubation at room temperature, the reaction was stopped by heating at 100°C and the incubation mixture evaporated to dryness in a heating block (80%) under a stream of N,. This was repeated at least 5 times with the addition of 1 ml of water each time. Aliquots were then counted in a liquid scintilla- tion counter. In addition, in the case of alanine and glycine, a portion of the reaction mixture was chro- matographed on a silica gel plate (Eastman Kodak Co., n-propyl alcohol/H,O, 7:3) and the substrate re- gion (marked by ninhydrin-stained guide strips) was eluted for a further determination of radioactivity and concentration of compound. Values for glycine and alanine shown were based on samples so recovered by chromatography, but were similar when measured after evaporation of water alone. Controls for each substrate included identical incubation without enzyme and with heat-inactivated enzyme. Qualitatively similar findings were obtained for all compounds when incubated with 2 units of enzyme.

* Complete equilibration of one substrate hydrogen with water was calculated to represent a specific activity of 6 x lo5 dpm/smole.

c With this compound only, added concentration was 0.02 M.

* In this experiment, the reaction was stopped by bringing the incubation mixture to 1 N HCl at room temperature; repeated evaporation was carried out at room temperature in a vacuum dessicator.

propionate, 2-bromopropionate, N-acetyl- L-alanine, and @-chloro-L-alanine. These compounds were selected for such a test partly because of their relatively high binding affinity, as estimated by Ki values. r.-Alanine was included as a check on the method. The data of Table III show that alanine had exchanged about 15% of one hydrogen (presumably the a-H), that gly- tine is also a substrate by this criterion, and that in none of the other compounds tested was significant hydrogen exchange catalyzed by the enzyme. @-Chloroalanine became significantly labeled during incubations, but this reaction was independent of enzyme. It is of interest that about the same extent of 3H-incorporation into gly- tine and alanine occurred with two differ- ent enzyme levels, one threefold greater than the other.

DISCUSSION

Some of the compounds tested in the work reported here have been examined earlier with alanine racemase from other sources, in some cases with results that differ from ours. Thus it was earlier reported that the alanine racemase of Lac- tobacillus fermenti is activated by acetate (4) while we find that acetate, and a group of other short-chain fatty acids, are inhibitors. In the same study (4) it was also reported that L-cysteine, L-serine, L-alaninol, and L-cy-amino-n-butyrate were more or less inhibitory while glycine was not. We confirm the interesting finding of Lambert and Neuhaus (5) that m-l-aminoethylphosphonate is a potent inhibitor of the enzyme.

The data of Table II permit some unexpected generalizations about the structural requirements for binding. An a-amino group is not essential for binding and when present does not add to the binding. At equal concentration, acetate inhibits slightly more than glycine while the Ki of propionate is close to the K, for L- or

n-alanine. Acrylate binds considerably bet- ter than does alanine, possibly because its planar C-cu geometry resembles that of the transition state for alanine racemization. Inhibition of proline racemase by the pla-


nar pyrrole-2-carboxylate (9) was an early example of the relatively tight binding of transition-state analogs (13).

The presence of the a-amino group does not enhance binding nor does it reduce binding even when its electronic character and bulk are drastically changed as in acetyl-L-alanine, L-alanyl-L-alanine, P-alanyl-L-alanine, t-aminocaproyl-L-alanine, and L-lysyl-L-alanine, all of which bind about as well as alanine itself. This indif- ference to the a-amino group appeared sur- prising to us for a pyridoxal phosphate enzyme; however, it has been pointed out to us that our observations are paralleled by the relatively tight binding to arginine de- carboxylase of a-halo and cr-hydroxy analogs (14).

Substituents on the P-carbon of alanine appear generally to reduce binding, independent of their chemical nature, e.g., 2,3-diaminopropionate and cysteine, as well as serine and other noninhibitory amino acids. The same reduction in binding is seen when a ,L?-substituent is added to propionate (@-alanine, butyrate), although 3-chloropropionate and valerate are bound nearly as well as propionate. Substitution at the a-carbon of alanine, based on the single case of cY-aminoisobutyrate, completely destroys binding. Substi- tution of a halo or hydroxyl group for the a-amino group of alanine (2-bromopropionate, lactate) still permits binding, as might be expected from the dispensability of the amino group itself. Absence of the carboxylate anion (alanine methyl ester, alaninol) was compatible with weak binding.

The singular potency of l-aminoethylphosphonate as an inhibitor is not readily rationalized by our data, although a possible correlate is the acid strength of the phosphonic acid group relative to the car- boxyl. Its striking contrast with the noninhibitory 2-aminoethylphosphonate, however, is consistent with the generalization concerning the effect of P-substitution on alanine or propionate and points up the analogy between propionate and ethylphosphonate.

It should be noted that an observation

noted under Kinetic Constants, failure of a-H substitution by deuterium to influence K, while substantially decreasing V, sug- gests that K, is to a good approximation a true binding constant and can be directly compared with the Ki values discussed above.

Several halo analogs (2-bromopropionate, 3-chloropropionate) were tested with the possibility that a sulfhydryl at the active site (inferred from the marked inhibition by mercuribenzoate) might be cova- lently derivatized by such compounds. No evidence was noted, however, for a time- dependent, irreversible inhibition of the enzyme by those compounds under the single set of conditions used.

Of seven compounds tested for substrate activity by incubation with the racemase in 3H20, only alanine and glycine showed unequivocal enzyme-dependent incorporation of 3H. Even after long incubation with an amount of enzyme that should have completely exchanged a-‘H with solvent 3H, only about 15% of the expected activity was found in the alanine. In addition, tri- pling the quantity of enzyme failed to yield clearly greater incorporation, an observation which argues against a kinetic isotope effect. The basis for incomplete equilibration, more clearly demonstrated here than previously (11, 12), is obscure and will require further study.

Although glycine showed considerable apparent incorporation of solvent 3H with inactive enzyme, the incubation mixture containing active enzyme gave 20-fold greater incorporation. Presumably, both enantiotopic hydrogens of glycine exchange; it would be of interest to deter- mine whether both are labilized at the same rate.

The substrate status of P-chloroalanine could not be settled. This compound, if a substrate, would offer the advantage of a sufficiently high optical rotation to make a continuous polarimetric assay of the enzyme more feasible than with alanine as substrate. However, polarimetric trials showed no enzymatic racemization, and the data of Table III show no increment of enzymatic exchange above the considera-


ble non-enzymatic exchange observed. While the high rate of nonenzymatic exchange could be reduced by avoiding the final heating step, it was still large.

Finally, the kinetic data in both direc- tions, in qualitative agreement with such data for the alanine racemase from other sources (4, 5), indicate rather similar K, and V values for both L- and n-alanine. Studies now in progress will show whether this kinetic symmetry also applies to the rates of protonation and deprotonation of the presumed intermediate, the pyridoxal phosphate-stabilized C-cu anion, a question discussed in (15).

REFERENCES

1. ADAMS, E. (1972), in The Enzymes (P. D. Boyer, ed.), Vol. 6, pp. 479-507, 3rd ed., Academic Press, Inc., New York.

2. FREE, C. A., MARC, J., ARNOW, P., AND BARRY, G. T. (1967) Biochim. Biophys. Acta 146.608-610.

3. Rosso, G., TAKASHIMA, K., AND ADAMS, E. (1969) Biochem. Biophys. Res. Commun. 34,134-140.

4. JOHNSTON, M M., ANDDIVEN, W. F. (1969) J. Biol. Chem. 244, 5414-5420.

5. LAMBERT, M. P., AND NEUHAUS, F. C. (1972) J. Bacterial. 110, 978-987.

6. FISCHER, E., AND RASKE, K. (1907) Ber. Deut. Chem. Ges. 40, 3717-3724.

7. EBATA, M. (1959) J. Biochem. 46, 397-406. 8. ERLANGER, B. F., AND BRAND, E. (1951) J. Amer.

Chem. Sot. 73, 4025-4027. 9. CARDINALE, G. J., AND ABELES, R. H. (1968)

Biochemistry 7,3970-3978. 10. FINLAY, T. H., AND ADAMS, E. (1970) J. Biol.

Chem. 245, 5348-5760. 11. GLASER, L. (1960) J. Biol. Chem. 235,2095-2098. 12. JOHNSTON, R. B., SCHOLZ, J. J., DIVEN, W. F., AND

SHEPERD, S. (1968) in Pyridoxal Catalysis: En- zymes and Model Systems (E. E. Snell et al, ed.), p. 537, Wiley Interscience, New York.

13. LIENHARD, G. (1973) Science 180, 149-154. 14. BLETHEN, S. L., BOEKER, E. A., and SNELI, E. E.

(1968) J. Biol. Chem. 243, 1671-1677. 15. DUNATHAN, H. C. (1971) A&an. Enzymol. 35,

79-134.

alanine racemase of pseudomonas: observations on subtrate and inhibitor specificity

Documents