Racemase Reaction Research Articles

A Novel Bifunctional Amino Acid Racemase With Multiple Substrate Specificity, MalY From Lactobacillus sakei LT-13: Genome-Based Identification and Enzymological Characterization.

The Lactobacillus sakei strain LK-145 isolated from Moto, a starter of sake, produces potentially large amounts of three D-amino acids, D-Ala, D-Glu, and D-Asp, in a medium containing amylase-digested rice as a carbon source. The comparison of metabolic pathways deduced from the complete genome sequence of strain LK-145 to the type culture strain of Lactobacillus sakei strain LT-13 showed that the L- and D-amino acid metabolic pathways are similar between the two strains. However, a marked difference was observed in the putative cysteine/methionine metabolic pathways of strain LK-145 and LT-13. The cystathionine β-lyase homolog gene malY was annotated only in the genome of strain LT-13. Cystathionine β-lyase is an important enzyme in the cysteine/methionine metabolic pathway that catalyzes the conversion of L-cystathionine into L-homocysteine. In addition to malY, most genome-sequenced strains of L. sakei including LT-13 lacked the homologous genes encoding other putative enzymes in this pathway. Accordingly, the cysteine/methionine metabolic pathway likely does not function well in almost all strains of L. sakei. We succeeded in cloning and expressing the malY gene from strain LT-13 (Ls-malY) in the cells of Escherichia coli BL21 (DE3) and characterized the enzymological properties of Ls-MalY. Spectral analysis of purified Ls-MalY showed that Ls-MalY contained a pyridoxal 5′-phosphate (PLP) as a cofactor, and this observation agreed well with the prediction based on its primary structure. Ls-MalY showed amino acid racemase activity and cystathionine β-lyase activity. Ls-MalY showed amino acid racemase activities in various amino acids, such as Ala, Arg, Asn, Glu, Gln, His, Leu, Lys, Met, Ser, Thr, Trp, and Val. Mutational analysis revealed that the 𝜀-amino group of Lys233 in the primary structure of Ls-MalY likely bound to PLP, and Lys233 was an essential residue for Ls-MalY to catalyze both the amino acid racemase and β-lyase reactions. In addition, Tyr123 was a catalytic residue in the amino acid racemase reaction but strongly affected β-lyase activity. These results showed that Ls-MalY is a novel bifunctional amino acid racemase with multiple substrate specificity; both the amino acid racemase and β-lyase reactions of Ls-MalY were catalyzed at the same active site.

A sensitive assay for measuring aspartate-specific amino acid racemase activity

d-Aspartate (d-Asp), a free d-amino acid found in mammals, plays crucial roles in the central nervous, neuroendocrine, and endocrine systems. In mammalian tissues, d-Asp oxidase (DDO) is a degradative enzyme that stereospecifically acts on d-Asp. Asp racemase, a synthetic enzyme that produces d-Asp from l-Asp, has been identified in several lower organisms; however, the biosynthetic pathway of d-Asp in mammals remains to be fully clarified. The aim of this study was to establish a simple, accurate, and sensitive enzymatic method for the determination of Asp racemase activity. Using recombinant Streptococcus thermophilus Asp racemase as a model enzyme, two enzymatic methods for the determination of Asp racemase activity were optimized. In these methods, recombinant human DDO was used to degrade d-Asp formed from l-Asp by the Asp racemase reaction to 2-oxo acid, the amounts of which were then determined using a colorimetric assay. In one method, designated the coupling method, DDO was concomitantly included in the Asp racemase reaction mixture, and the Asp racemase reaction was readily coupled to the d-Asp degradative reaction by DDO during the incubation. In the other method, designated the separating method, an aliquot of the Asp racemase reaction mixture was mixed with DDO after the reaction to determine the amounts of d-Asp produced by Asp racemase. Under optimized conditions, the accuracy and sensitivity of these two methods were examined and compared, both to one another and conventional high-performance liquid chromatography (HPLC). The results presented here suggest that the coupling method is more accurate and sensitive than the other two methods and can be used for the determination of Asp racemase activity. The coupling method may help to advance our current understanding of the biosynthetic pathway of d-Asp in mammals.

Site‐Directed Mutagenesis of Rice Serine Racemase: Evidence That Glu219 and Asp225 Mediate the Effects of Mg2+ on the Activity

We succeeded in constructing the Glu219Ala/Asp225Ala (i.e., E219A/D225A) serine racemase (SerR) by site-directed mutagenesis, and the effects of Mg(2+) on the catalytic efficiency and the structure were compared between the E219A/D225A-SerR and the wild-type protein. This is the first example of a serine racemase whose amino acid residues in the Mg(2+)-binding site were replaced with other amino acids by site-directed mutagenesis. Neither the serine racemase nor the dehydratase activities of the E219A/D225A-SerR were affected by the addition of Mg(2+), and Glu219 and Asp225 of the SerR are the essential amino acid residues for Mg(2+) to affect both kinds of enzyme activities. Therefore, Glu219 and Asp225 mediate the effects of Mg(2+) on the activity and are important for the SerR to form the Mg(2+)-binding site. Judging from the difference of the K(eq) values between the E219A/D225A-SerR and the SerR, Mg(2+) might affect the equilibrium states in the racemase reaction. The fluorescence quenching analysis of the E219A/D225A-SerR showed that Mg(2+) bound to Glu219 and Asp225 of the SerR probably causes a conformational change in the ternary structure of the SerR.

Occurrence of D-serine in rice and characterization of rice serine racemase

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene ( serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg 2+ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg 2+, and this structural change probably regulates the two enzymatic activities.

Compound-Specific Nitrogen Isotope Analysis of d-Alanine, l-Alanine, and Valine: Application of Diastereomer Separation to δ15N and Microbial Peptidoglycan Studies

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

Search for Extraterrestrial Life Using Chiral Molecules: Mandelate Racemase as a Test Case

We have investigated an enzymatic racemization reaction as a marker for extraterrestrial life, which resulted in a change in optical rotation of a mandelic acid over time, as measured by polarimetry. Mandelate racemase was active in aqueous buffer in a temperature range between 0 degrees C and 70 degrees C and also in concentrated ammonium salt solutions and water-in-oil microemulsions in a temperature range between -30 degrees C and 60-70 degrees C; however, the enzyme was not active in several organic cryosolvents. Thus, we have demonstrated that concentrated ammonium salt solutions and water-in-oil microemulsions, both of which are able to form on extraterrestrial planets and moons in the presence of liquid water, are suitable media for enzyme reactions at subzero temperatures. Kinetic data for the mandelate racemase reaction obtained by polarimetry, while reproducible and internally consistent, differed significantly from several sets of data obtained previously by other methods such as chromatography and hydrogen-deuterium exchange. However, we conclude that reactions yielding a polarimetric signal, such as the racemizations employed in this work, are suitable mechanisms by which to utilize a change in chirality over time as a tool to detect signs of life.

A novel assay method for an amino acid racemase reaction based on circular dichroism

We have established a novel assay method based on circular dichroism that can be used for the kinetic study of the activity of amino acid racemases, such as ALR (alanine racemase). Although an enzyme-coupled assay method has been used to measure racemase activity, the CD method is superior to the enzyme assay because it can accurately determine the immediate changes of an enantiomer on racemization between its L- and D-forms. The enzyme-coupled assay requires D-amino acid oxidase, which is inactivated by an inhibitor of ALR, D-cycloserine. This indicates that the inhibitory kinetic study for ALR with D-cycloserine by the enzyme-coupled assay method is restricted to the analysis of only the reaction resulting in the formation of L-Ala from D-Ala. However, since the CD assay does not require the coupled enzyme, it can be used to comprehensively evaluate the reactions that result in the formation both of D-Ala from L-Ala and of L-Ala from D-Ala at several substrate concentrations. Streptomyces ALR also catalyses the formation of D-Ser from L-Ser and of L-Ser from D-Ser, but the catalytic constants (kcat) are 4- and 10-fold lower than those for the formation of D-Ala from L-Ala and of L-Ala from D-Ala respectively.

Serine Racemase Modulates Intracellular D-Serine Levels through an α,β-Elimination Activity

Mammalian brain contains high levels of d-serine, an endogenous co-agonist of N-methyl D-aspartate type of glutamate receptors. D-Serine is synthesized by serine racemase, a brain enriched enzyme converting L- to D-serine. Degradation of D-serine is achieved by D-amino acid oxidase, but this enzyme is not present in forebrain areas that are highly enriched in D-serine. We now report that serine racemase catalyzes the degradation of cellular D-serine itself, through the alpha,beta-elimination of water. The enzyme also catalyzes water alpha,beta-elimination with L-serine and L-threonine. alpha,beta-Elimination with these substrates is observed both in vitro and in vivo. To investigate further the role of alpha,beta-elimination in regulating cellular D-serine, we generated a serine racemase mutant displaying selective impairment of alpha,beta-elimination activity (Q155D). Levels of D-serine synthesized by the Q155D mutant are several-fold higher than the wild-type both in vitro and in vivo. This suggests that the alpha,beta-elimination reaction limits the achievable D-serine concentration in vivo. Additional mutants in vicinal residues (H152S, P153S, and N154F) similarly altered the partition between the alpha,beta-elimination and racemization reactions. alpha,beta-Elimination also competes with the reverse serine racemase reaction in vivo. Although the formation of L- from D-serine is readily detected in Q155D mutant-expressing cells incubated with physiological D-serine concentrations, reversal with wild-type serine racemase-expressing cells required much higher D-serine concentration. We propose that alpha,beta-elimination provides a novel mechanism for regulating intracellular D-serine levels, especially in brain areas that do not possess D-amino acid oxidase activity. Extracellular D-serine is more stable toward alpha,beta-elimination, likely due to physical separation from serine racemase and its elimination activity.

Evolution of Enzymatic Activity in the Enolase Superfamily: Structural Studies of the Promiscuous o-Succinylbenzoate Synthase from Amycolatopsis,

Divergent evolution of enzyme function is commonly explained by a gene duplication event followed by mutational changes that allow the protein encoded by the copy to acquire a new function. An alternate hypothesis is that this process is facilitated when the progenitor enzyme acquires a second function while maintaining the original activity. This phenomenon has been suggested to occur in the o-succinylbenzoate synthase (OSBS) from a species of Amycolatopsis that catalyzes not only the physiological syn-dehydration reaction of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate but also an accidental racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. To understand the molecular basis of this promiscuity, three-dimensional structures of liganded complexes of this enzyme have been determined, including the product of the OSBS reaction and three N-acylamino acid substrates for the N-acylamino acid racemase (NAAAR) reaction, N-acetylmethionine, N-succinylmethionine, and N-succinylphenylglycine, to 2.2, 2.3, 2.1, and 1.9 A resolution, respectively. These structures show how the active-site cavity can accommodate both the hydrophobic substrate for the OSBS reaction and the substrates for the accidental NAAAR reaction. As expected, the N-acylamino acid is sandwiched between lysines 163 and 263, which function as the catalytic bases for the abstraction of the alpha-proton in the (R)- and (S)-racemization reactions, respectively [Taylor Ringia, E. A., Garrett, J. B, Thoden, J. B., Holden, H. M., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 42, 224-229]. Importantly, the protein forms specific favorable interactions with the hydrophobic amino acid side chain, alpha-carbon, carboxylate, and the polar components of the N-acyl linkage. Accommodation of the components of the N-acyl linkage appears to be the reason that this enzyme is capable of a racemization reaction on these substrates, whereas the orthologous OSBS from Escherichia coli lacks this functionality.

Assessing the Reversibility of the Anaplerotic Reactions of the Propionyl-CoA Pathway in Heart and Liver

While a number of studies underline the importance of anaplerotic pathways for hepatic biosynthetic functions and cardiac contractile activity, much remains to be learned about the sites and regulation of anaplerosis in these tissues. As part of a study on the regulation of anaplerosis from propionyl-CoA precursors in rat livers and hearts, we investigated the degree of reversibility of the reactions of the propionyl-CoA pathway. Label was introduced into the pathway via NaH13CO3, [U-13C3]propionate, or [U-13C3]lactate + [U-13C3]pyruvate, under various concentrations of propionate. The mass isotopomer distributions of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA revealed that, in intact livers and hearts, (i) the propionyl-CoA carboxylase reaction is slightly reversible only at low propionyl-CoA flux, (ii) the methylmalonyl-CoA racemase reaction keeps the methylmalonyl-CoA enantiomers in isotopic equilibrium under all conditions tested, and (iii) the methylmalonyl-CoA mutase reaction is reversible, but its reversibility decreases as the flow of propionyl-CoA increases. The thermodynamic dis-equilibrium of the combined reactions of the propionyl-CoA pathway explains the effectiveness of anaplerosis from propionyl-CoA precursors such as heptanoate.

Molecular dynamics studies of alanine racemase: a structural model for drug design.

Alanine racemase (AlaR) is a bacterial enzyme that catalyzes the interconversion of L- and D-alanine, which is an essential constituent of the peptidoglycan layer of the bacterial cell wall and requires pyridoxal 5'-phosphate (PLP) as a cofactor. The enzyme is universal to bacteria, including mycobacteria, making it an attractive target for drug design. To investigate the effects of flexibility on the binding modes of the substrate and an inhibitor and to analyze how the active site is affected by the presence of the substrate versus inhibitor, a molecular dynamics simulation on the full AlaR dimer from Bacillus stearothermophilus (pdb code: 1SFT) with a D-alanine molecule in one active site and the noncovalent inhibitor, propionate, in the second site has been carried out. Within the time scale of the simulation, we show that the active site becomes more stabilized in the presence of substrate versus inhibitor. The results of this simulation are in agreement with the proposed mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by acting cooperatively with Tyr 265' and Lys 39. A structural water molecule in contact with both substrate and inhibitor (i.e., in both active sites) and bridging residues in both active sites was identified. It shows a remarkably low mobility and does not exchange with bulk water. This water molecule can be taken into account for the design of specific AlaR inhibitors by either utilizing it as a bridging group or displacing it with an inhibitor atom. The results presented here provide insights into the dynamics of the alanine racemase in the presence of substrate/inhibitor, which will be used for the rational design of novel inhibitors.

Conversion of a PLP-dependent racemase into an aldolase by a single active site mutation.

Alanine racemase (Alr) [EC 5.1.1.1] from Geobacillus stearothermophilus is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the first committed step in bacterial cell wall biosynthesis. It is converted to an aldolase upon replacement of Tyr265, which normally serves as a catalytic base in the racemase reaction, with alanine. The Y265A mutation increases catalytic efficiency for cleavage of beta-phenylserine to benzaldehyde and glycine by 2.3 x 105 fold as compared to the wild-type racemase, while racemase activity is greatly decreased. Additional mutagenesis suggests that His166 may act as the base that initiates the retroaldol reaction. The Y265A mutant is highly stereoselective for (2R,3S)-phenylserine, a d-amino acid, and does not process its enantiomer. This preference is consistent with the expected binding mode of substrate in the modified active site and supports the proposal that naturally occurring d-threonine aldolases and alanine racemases derive from a common ancestor.

Occurrence of a unique amino acid racemase in a basidiomycetous mushroom, Lentinus edodes

Free d-alanine was detected in a cell extract of the fruit-body of an edible basidiomycetous mushroom, Lentinus edodes (Shiitake), by means of reverse-phase high performance liquid chromatography. We also found an amino acid racemase activity in L. edodes fruit-body, and purified the enzyme. The enzyme has a molecular weight of approximately 86,000, and consists of two subunits of identical molecular weight (44,000). The optimal pH of the enzyme activity is around pH 9.5 for both d-to- l and l-to- d alanine racemization. The enzyme requires pyridoxal 5′-phosphate as a cofactor. K m and V max values for d-alanine were 37.3 mM and 520 nmol/min/mg, respectively; for l-alanine, they were 9.21 mM and 141 nmol/min/mg, respectively. The equilibrium constant was calculated to be 1.10, which is consistent with the theoretical value for the racemase reaction. The ability of the enzyme to catalyze the racemization of various d-amino acids was investigated. The enzyme catalyzes the racemization of d-serine (relative reaction rate, 144% of rate for d-alanine), d-alanine (100%), d-homoserine (17.1%), d-2-aminobutyrate (5.6%), d-glutamate (4.5%), and d-asparagine (3.2%). To the best of our knowledge, this is the first report of an amino acid racemase produced by a basidiomycetous mushroom.

Reaction Mechanism of Alanine Racemase from Bacillus stearothermophilus: X-RAY CRYSTALLOGRAPHIC STUDIES OF THE ENZYME BOUND WITH N-(5′-PHOSPHOPYRIDOXYL)ALANINE

The crystal structures of alanine racemase bound with reaction intermediate analogs, N-(5'-phosphopyridoxyl)-L-alanine (PLP-L-Ala) and N-(5'-phosphopyridoxyl)-D-alanine (PLP-D-Ala), were determined at 2.0-A resolution with the crystallographic R factor of 17.2 for PLP-L-Ala and 16.9 for PLP-D-Ala complexes. They were quite similar not only to each other but also to the structure of the native pyridoxal 5'-phosphate (PLP)-form enzyme; root mean square deviations at Calpha among the three structures were less than 0.28 A. The side chains of the amino acid residues around the PLP-L-Ala and PLP-D-Ala were virtually superimposable on each other as well as on those around PLP of the native holoenzyme. The alpha-hydrogen of the alanine moiety of PLP-L-Ala was located near the OH of Tyr(265)', whereas that of PLP-D-Ala was near the NZ of Lys(39). These support the previous findings that Tyr(265)' and Lys(39) are the catalytic bases removing alpha-hydrogen from L- and D-alanine, respectively. The prerequisite for this two-base mechanism is that the alpha-proton abstracted from the substrate is transferred (directly or indirectly) between the NZ of Lys(39) and the OH of Tyr(265'); otherwise the enzyme reaction stops after a single turnover. Only the carboxylate oxygen atom of either PLP-Ala enantiomer occurred at a reasonable position that can mediate the proton transfer; neither the amino acid side chains nor the water molecules were located in the vicinity. Therefore, we propose a mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by mediating the proton transfer between the two catalytic bases, Lys(39) and Tyr(265)'. The results of molecular orbital calculation also support this mechanism.

Reaction mechanism of gramicidin S synthetase 1, phenylalanine racemase, of Bacillus brevis.

We have demonstrated that gramicidin S synthetase 1 (GS 1), phenylalanine racemase [EC 5.1.1.11], of Bacillus brevis catalyzes the exchange between a proton in the medium and alpha-hydrogen of phenylalanine in the course of the racemase reaction by using tritiated water or L-phenyl[2,3-3H]alanine. GS 1 from some gramicidin S non-producing mutants of B. brevis lacking phenylalanine racemase activity did not catalyze the tritium exchange reaction. The proton exchange between phenylalanine bound as thioester on the GS 1-phenylalanine complex and water in the medium was detected, but 5,5'-dithiobis(2-nitrobenzoic acid)-modified complex lacked both the proton exchange and phenylalanine racemase activity. It is suggested that a base group, probably a sulfhydryl group, on the enzyme functions as proton donor and acceptor during the phenylalanine racemase reaction.

Distribution of glutamate racemase in lactic acid bacteria and further characterization of the enzyme from Pediococcus pentosaceus.

We investigated the distribution of glutamate racemase (EC 5.1.1.3) in bacteria, and found that the enzyme occurs exclusively in lactic acid bacteria. Pediococcus pentosaceus IFO 3182 produces the enzyme most abundantly. The enzyme purified from extracts of the Escherichia coli clone cells carrying the plasmid pICR221, which contains the enzyme gene of P. pentosaceus, does not require cofactors for its catalytic activity [N. Nakajima, K. Tanizawa, H. Tanaka and K. Soda, Agric. Biol. Chem., 50, 2823 (1986)]. This cofactor dispensability was re-confirmed with the enzyme purified from cell extracts of P. pentosaceus. On the basis of kinetic parameters obtained by measurements of the initial velocities, the glutamate racemase reaction was categorized into a typical racemization with a calculated equilibrium constant of 1.05. D- and L-Glutamate were the specific substrates for the enzyme, although L-homocysteinesulfinate, a sulfur analog of L-glutamate, slightly served as a substrate. L-α-Aminoadipate behaved as a competitive inhibitor in the racemization of glutamate. The enzyme was inactivated significantly by treatment with various thiol-blocking reagents.

A single assay for simultaneously testing effectors of alanine racemase and/or D-alanine: D-alanine ligase.

The biosynthesis from L-alanine of D-alanyl-D-alanine, required for the peptidoglycan layer of the cell wall of many bacterial species, is catalyzed by two enzymes in series, alanine racemase and D-alanine: D-alanine ligase. A simple in vitro method, called the combined assay, for simultaneously testing for effectors of either or both enzymes in a single assay by coupling these enzymes to each other is described here. The experiments used to derive the optimum conditions for the assay are also described. Each enzyme is included in the assay in rate-limiting amounts, wherein the product of the initial racemase reaction, D-alanine, becomes the substrate for the subsequent ligase. The product of the overall reaction, [14C]-D-alanyl-D-alanine, is separated chromatographically from the L-[1-14C]alanine substrate, and from any D-[1-14C]alanine intermediate, at the end of the incubation, is counted and the percent conversion of substrate to product calculated. The inhibitory effects of 3-fluoro-D-alanine-2d, a known inhibitor of the racemase, and D-cycloserine and DL-1-aminoethylphosphonic acid, inhibitors of both enzymes, were readily detectable. The sensitivity of the combined assay to these inhibitors appears similar to that of earlier assays. This assay has the advantage over previous ones of being able to detect inhibitors of either enzyme in a single assay, thereby avoiding the need to screen each compound in a separate assay of each enzyme.

A birect kinetic assay for mandelate racemase using circular dichroic measurements

A new, direct kinetic assay for mandelate racemase has been developed. The assay depends upon changes with time in circular dichroic ellipticities of mandelate solutions at 262 nm. The technique should allow more precise kinetic measurements of the mandelate racemase reaction than have heretofore been possible. Also, the approach to equilibrium can be measured from either ( R)- or ( S)-mandelate with equal facility. The technique, should, moreover, be extendible to other enzyme-catalyzed reactions involving changes with time in circular dichroic ellipticities.

De novo synthesis of d-alanine in germinating Pisum sativum seedlings

The amounts of d-alanine derivatives, γ- l-glutamyl- d-alanine and N-malonyl- d-alanine, increase rapidly during the early growth of pea seeds. Pyruvate-[1− 14C], l-alanine-[U− 14C], d-alanine-[U− 14C], l-alanine-[ 15N] and 15NH 4Cl were therefore fed to the seedlings and the incorporation investigated. Labelling results revealed that pea seedlings can utilize these erogenous compounds to form d-alanine and that labelled l-alanine is effectively converted to the d-enantiomer with retention of 14C and, largely, 15N label. Enzyme analyses in vitro provided additional evidence that the extract of pea seedlings catalyzes the direct conversion of l-alanine to d-alanine. The data suggest that the de novo synthesis of d-alanine in pea seedlings occurs by a racemase reaction.