Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity*

Young Hee LimDagger , Tohru YoshimuraDagger , Yoichi KurokawaDagger , Nobuyoshi EsakiDagger §, and Kenji Soda

From the Dagger  Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611 and  Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35, Yamate-Cho, Suita, Osaka 564, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Pyridoxal 5'-phosphate-dependent amino acid racemases of broad substrate specificity catalyze transamination as a side reaction. We studied the stereospecificities for hydrogen abstraction from C-4' of the bound pyridoxamine 5'-phosphate during transamination from pyridoxamine 5'-phosphate to pyruvate catalyzed by three amino acid racemases of broad substrate specificity. When the enzymes were incubated with (4'S)- or (4'R)-[4'-3H]pyridoxamine 5'-phosphate in the presence of pyruvate, tritium was released into the solvent from both pyridoxamine 5'-phosphates. Thus, these enzymes abstract a hydrogen nonstereospecifically from C-4' of the coenzyme in contrast to the other pyridoxal 5'-phosphate-dependent enzymes so far studied, which catalyze the stereospecific hydrogen removal. Amino acid racemase of broad substrate specificity from Pseudomonas putida produced D- and L-glutamate from alpha -ketoglutarate through the transamination with L-ornithine. Because glutamate does not serve as a substrate for racemization, the enzyme catalyzed the nonstereospecific overall transamination between L-ornithine and alpha -ketoglutarate. The cleavage and formation of the C-H bond at C-4' of the coenzyme and C-2 of the substrate thus occurs nonstereospecifically on both sides of the plane of the coenzyme-substrate complex intermediate. Amino acid racemase of broad substrate specificity is the first example of a pyridoxal enzyme catalyzing nonstereospecific transamination.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Although enzymatic racemization of amino acid is apparently simple, consisting of a nonstereospecific rearrangement of the substrate alpha -hydrogen, several different types of amino acid racemases are found in microorganisms. Aspartate racemase (EC 5.1.1.13) (1) and glutamate racemase (EC 5.1.1.3) (2, 3) are independent of cofactors, and one or more cysteinyl residues play an important role in the abstraction of the alpha -hydrogen from the substrate. Phenylalanine racemase (EC 5.1.1.11), which is involved in gramicidin S synthesis, utilizes ATP (4). Alanine racemase (EC 5.1.1.1) in several microorganisms (5), arginine racemase (EC 5.1.1.9) from Pseudomonas graveolens (6), and amino acid racemases of broad substrate specificity (EC 5.1.1.10) of Aeromonas punctata (7), Pseudomonas striata (8), and Pseudomonas putida (9) all depend on pyridoxal 5'-phosphate (PLP)1.

Faraci and Walsh (10) proposed a mechanism for alanine racemase that is probably similar to that of other PLP-dependent amino acid racemases. The reaction is initiated by transaldimination. In this step, PLP bound with the active-site lysyl residue through an internal Schiff base (Scheme IA) reacts with a substrate to form an external Schiff base (B). The subsequent alpha -hydrogen abstraction results in the formation of a resonance-stable anionic intermediate (C). If the reprotonation occurs at C-2 of the substrate moiety on the opposite face of the planar intermediate to that where the proton abstraction occurs, an antipodal aldimine is formed (D). The aldimine complex is subsequently hydrolyzed to form isomerized amino acid and regenerates the bound PLP (E).


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Scheme I.   Reaction mechanism of amino acid racemase.

The random return of hydrogen to the anionic intermediate is a characteristic of enzymatic racemization among various pyridoxal enzyme reactions. In aminotransferase reactions, the abstracted hydrogen is stereospecifically transferred to C-4' of the cofactor, and a ketimine intermediate is formed (11). The pyridoxamine 5'-phosphate (PMP) form of the enzyme and a keto acid are produced by hydrolysis of the ketimine intermediate. Amino acid racemases are reported to catalyze the transamination as a side reaction (12). The transamination catalyzed by amino acid racemases can be attained through a sequence either A right-arrow B right-arrow F (or G) right-arrow H or A right-arrow B right-arrow C right-arrow F (or G) right-arrow H (Scheme I). An equivalent route can be delineated for the antipode: E right-arrow D right-arrow F (or G) right-arrow H or E right-arrow D right-arrow C right-arrow F (or G) right-arrow H.

In transamination, mutual hydrogen transfer between the substrate and C-4' of the cofactor occurs. In all previous studies of transaminations catalyzed by aminotransferases such as L-aspartate aminotransferase (AspAT) (13), L-alanine aminotransferase (14), dialkylamino acid aminotransferase (15), pyridoxamine:pyruvate aminotransferase (16), D-amino acid aminotransferase (D-AAT) (17), and branched chain L-amino acid aminotransferase (BCAT) (17) as well as other pyridoxal enzymes such as L-serine hydroxymethyltransferase (18), L-tryptophan synthase (19), L-glutamate decarboxylase (20), and L-aspartate beta -decarboxylase (21), the hydrogen transfer between substrate and cofactor occurs strictly stereospecifically on the si or re face of the plane of the anionic intermediate. However, if the transamination catalyzed by amino acid racemases proceeds as depicted in Scheme I, the hydrogen transfer should occur nonstereospecifically on both faces of the planar intermediate. We were therefore interested in the stereospecificity of the hydrogen transfer during transamination catalyzed by amino acid racemases as a side reaction.

We provide here the first evidence that hydrogen removal from C-4' of PMP occurs randomly on both faces of the substrate-cofactor imine plane during half transamination catalyzed by the amino acid racemases. We also show that the enzyme catalyzes nonstereospecific overall transamination between L-ornithine and alpha -ketoglutarate as well.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Amino acid racemases with low substrate specificity from P. putida (9), P. striata (8), and A. punctata (7) were purified as described previously. BCAT of Escherichia coli K-12 (22) was kindly supplied by Professor H. Kagamiyama and Dr. K. Inoue of Osaka Medical College, Takatsuki, Japan. AspAT from pig heart was obtained from Boehringer Mannheim (Germany), 3H2O (3.7 GBq/g) was from DuPont. The other chemicals were of analytical grade.

Enzyme and Protein Assays-- The activity of the amino acid racemases was determined by measurement of a change in optical rotation at 365 nm with a Perkin-Elmer 241 polarimeter and also by a coupled assay procedure with D- or L-amino acid oxidase (9). Protein concentrations were determined by dye staining with a Bio-Rad protein assay reagent with bovine serum albumin as a standard.

Preparation of Apoenzyme-- Apo-amino acid racemases were prepared as described previously (7). An appropriate amount of enzyme (1-3 mg) was dialyzed against 10 mM potassium phosphate buffer (pH 8.0) containing 30 mM hydroxylamine and 0.1% 2-mercaptoethanol for 24 h and then dialyzed against 10 mM potassium phosphate buffer (pH 8.0) at 4 °C overnight. The apoenzyme formed was determined by measurement of the activity of the enzyme in the presence or absence of 10 mM PLP. Apo-enzymes recovered about 80% of their initial activities on addition of PLP. Apo-AspAT, apo-D-AAT, and apo-BCAT were prepared as described previously (17).

Spectrophotometric Measurements-- Spectrophotometric measurements were made with a Shimadzu UV-visible recording spectrophotometer UV-260 with a 1.0-cm light path at 25 °C.

Preparation of (4'S)- and (4'R)-[4'3H]PMP-- (4'S)- and (4'R)-[4'-3H]PMP were prepared by incubation of the randomly labeled [4'-3H]PMP with apo-BCAT and apo-AspAT, respectively, as described previously (17). The specific radioactivities of (4'S)- and (4'R)-[4'3H]PMP prepared were 1.54 × 106 and 1.35 × 106 dpm/µmol, respectively. Stereospecificities for labeling were confirmed by measurement of tritium liberation from both PMPs catalyzed by apo-BCAT and apo-AspAT in the presence of alpha -ketoglutarate as described previously (17).

Abstraction of C-4' Hydrogen of PMP during Transamination-- The reaction mixture (100 µl) contained 10 µmol of potassium phosphate buffer (pH 8.0), 5 nmol of sodium pyruvate, 1 nmol of (4'S)- or (4'R)-[4'-3H]PMP, and 5 nmol of each apo-amino acid racemase. The reaction was carried out at 30 °C for 3 h and terminated by the addition of 100 µl of 1 M HCl. The mixture was immediately frozen in liquid nitrogen and dried with a Speed Vac concentrator. The residue was dissolved with 200 µl of H2O and subjected to a radioactivity assay. The tritium released from PMP was expressed as volatile radioactivity, which was estimated by subtraction of the radioactivity finally remaining from the radioactivity initially added to the reaction mixture. The reaction mixture (100 µl) with apo-D-AAT and apo-AspAT contained 10 µmol of Tris-HCl buffer (pH 8.0), 10 nmol of sodium alpha -ketoglutarate, 1.0 nmol of (4'S)- or (4'R)-[4'-3H]PMP, and 168 µg of apo-AspAT or 182 µg of apo-D-AAT, respectively. The reaction was carried out at 30 °C for 15 min and terminated by the addition of 100 µl of 1 M HCl. Other conditions were the same as those of the reactions with amino acid racemases. Radioactivity was determined with a Packard Tri-Carb scintillation spectrometer with Clear-solI (Nacalai Tesque, Japan) as a scintillator.

Transamination between L-Ornithine and alpha -Ketoglutarate Catalyzed by Amino Acid Racemase from P. putida-- The reaction mixture (1 ml) consisted of 10 µmol of L-ornithine, 10 µmol of alpha -ketoglutarate, 0.05 µmol of PLP, 50 µmol of Tricine buffer (pH 8.5), and 20 µg of enzyme. The reaction was carried out at 37 °C for 2 h and terminated by boiling. After centrifugation, amino acids in the supernatant solution were derivatized to diastereomers with Marfey's reagent (23) and analyzed by HPLC.

    RESULTS
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Introduction
Procedures
Results
Discussion
References

Transamination by Amino Acid Racemases-- When the amino acid racemase of broad substrate specificity from P. putida was incubated with L-alanine, the absorption at 420 nm derived from the internal Schiff base decreased, with a concomitant increase in the absorption at 330 nm (Fig. 1a). This suggests that the coenzyme form of the enzyme was converted from PLP to PMP during the racemization. The reversal from PMP to PLP also occurred on the basis of a spectral shift. The addition of pyruvate to the PMP form of enzyme, which was prepared by incubation of PMP with the apo-enzyme, led to a decrease in absorbance at 330 nm and an increase in that at 420 nm (Fig. 1b). Similar spectral shifts were observed in the reactions of the PMP form of the amino acid racemases from P. striata and A. punctata with pyruvate (data not shown). The results indicate that these amino acid racemases catalyze the transamination as a side reaction. Amino acid racemase of P. putida catalyzes the overall transamination between ornithine and pyruvate. The specific activity for the transamination was 0.26 units/mg (~10.7 min-1). The rate of transamination was lower than that of the racemization of ornithine by a factor of 1.1 × 104. However, the rate of enzymatic transamination was at least several orders of magnitude higher than that of the nonenzymatic transamination, which was lower than the minimum value for accurate determination, ~4.0 × 10-6 min-1.


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Fig. 1.   Change in the absorption spectrum of amino acid racemase by alanine and pyruvate. a, effect of L-alanine on the absorption spectrum of the amino acid racemase of broad substrate specificity from P. putida. Curve 1, PLP-form of enzyme (0.17 mg) in 1 ml of 10 mM potassium phosphate buffer (pH 8.0) at 25 °C. Curves 2 through 4 represent the absorption spectra of the enzyme at successive intervals of time after the addition of 10 µmol of L-alanine: curve 2, 30 min; curve 3, 60 min; curve 4, 100 min. b, effect of pyruvate on the absorption spectrum of the PMP form of amino acid racemase from P. putida. Curve 1, apoenzyme (0.15 mg) in 1 ml of 10 mM potassium phosphate buffer (pH 8.0) at 25 °C. Curves 2 through 5 show the absorption spectrum of the enzyme at successive intervals of time after the addition of 2 nmol of PMP and 5 nmol of pyruvate. Curve 2, 10 min; curve 3, 30 min; curve 4, 60 min; curve 5, 90 min.

Stereospecificity of Amino Acid Racemases for Hydrogen Abstraction from C-4' of PMP during Half-transamination-- When the PMP form of an enzyme is converted to the PLP form by transamination with an amino acceptor (keto acid), one of the two hydrogens at C-4' of PMP is usually transferred stereospecifically to Calpha of the keto acid (24). We studied the stereospecificity of amino acid racemases for hydrogen abstraction from C-4' of PMP using the method described previously (17). Stereospecificity is determined by measurement of the radioactivity of 3H released from the PMPs, which are stereospecifically tritiated at C-4'. Each 5 nmol of apo-amino acid racemase was incubated with 1 nmol of (4'S)- or (4'R)-[4'-3H]PMP and 5 nmol of sodium pyruvate. We deduce that PMP was completely converted to PLP, because the PMP form of the amino acid racemase from P. putida recovered 100% of the activity theoretically expected. As shown in Table I, tritium was released equally from both (4'S)- and (4'R)-[4'-3H]PMPs in the presence of amino acid racemases. The amount of tritium released from each PMP was about 50% that which initially existed. The control experiment was done with D-AAT and AspAT. As reported previously (17), D-AAT and AspAT catalyzed the stereospecific removal of tritium from (4'R)- or (4'S)-[4'3H]PMP, respectively, as shown in Table I. These results confirm the stereospecific tritium labeling of both PMPs. No tritium was released from each PMP in the absence of enzyme. Thus, the amino acid racemases catalyze the non-stereospecific abstraction of hydrogen from C-4' of PMP. They are the first class of pyridoxal enzyme catalyzing the hydrogen removal on both sides of the plane of a substrate-cofactor complex during transamination.

                              
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Table I
Release of 3H from [4'-3H]PMPs by amino acid racemases

Overall Transamination by Amino Acid Racemase from P. putida-- If the hydrogen is introduced nonspecifically to C-2 of the keto acid moiety of the anionic intermediate on both sides of the planar intermediate during the half reaction of transamination, racemic amino acid is formed from the keto acid (Scheme I; H right-arrow F (or G) right-arrow C right-arrow B right-arrow A or H right-arrow F (or G) right-arrow C right-arrow D right-arrow E). We studied the stereochemistry of glutamate formed from alpha -ketoglutarate by transamination with L-ornithine catalyzed by the amino acid racemase of P. putida. After the reaction, the products were derivatized to diastereomers with Marfey's reagent (23) and subjected to HPLC. As shown in Fig. 2, both enantiomers of glutamate and ornithine were found. The amino acid racemase from P. putida catalyzes the racemization of ornithine, but glutamate is inert as a substrate for the racemase reaction (9). Thus, both enantiomers of glutamate were directly formed by transamination, not by racemization of one enantiomer produced through transamination. Amino acid racemase from P. putida catalyzes the nonstereospecific overall transamination.


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Fig. 2.   Chiral analysis of the products formed in the reaction of L-ornithine and alpha -ketoglutarate catalyzed by amino acid racemase from P. putida. After the reaction products were derivatized to diastereomers with Marfey's reagent, they were subjected to HPLC analysis with a Puresil C18 column (Waters). HPLC was carried out with a linear gradient of 20-60% acetonitrile in 50 mM triethylamine phosphoric acid buffer (pH 2.8) at a flow rate of 1.0 ml/min.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We here show that nonstereospecific hydrogen abstraction from C-4' of PMP occurs in the transamination between PMP and pyruvate catalyzed by amino acid racemases with broad substrate specificity. The enzyme from P. putida also catalyzes nonstereospecific overall transamination between ornithine and alpha -ketoglutarate. These results indicate that the cleavage and formation of the C-H bonds at C-4' of the coenzyme occur on both sides of the plane of the anionic intermediate. This model is compatible with the proposed mechanism for racemization. The removal and return of alpha -hydrogen of substrate occur on both sides of the plane (Scheme I). It may be interesting to examine whether the removal of substrate alpha -hydrogen and the introduction of coenzyme C-4' hydrogen occur on the same side of the plane. The examination will be facilitated by analysis of the configuration of 3H-labeled PMP formed in 3H2O by transamination with either D or L enantiomer of an appropriate substrate. However, the amino acid used for this purpose needs to be inert as a substrate for racemization; otherwise, PMP will be also derived from the antipode of the substrate enantiomer added initially. Glutamate might be the only candidate substrate for this purpose; however, this is not the case. Glutamate is inert as an amino donor in transamination,2 although alpha -ketoglutarate serves as an amino acceptor as described above.

Two mechanisms have been proposed for enzymatic racemization: a two-base mechanism and a one-base mechanism (25, 26). In the two-base mechanism, two different bases participate in the catalysis; one abstracts the hydrogen from a substrate, and the other returns a hydrogen to the deprotonated intermediate. Glutamate racemase (27, 28), aspartate racemase (1), proline racemase (25, 30, 31), and diaminopimelate epimerase (32), which do not require cofactors, catalyze racemization by this mechanism. In the one-base mechanism, a single amino acid residue abstracts the alpha -hydrogen from a substrate and nonstereospecifically returns it to the anionic intermediate.

A swinging door motion has been proposed by Henderson and Johnston (33) as a model to fit the one-base mechanism; the plane of the substrate·PLP complex acts like a swinging door in order that the base can be located on both faces of the plane. If only a single base is involved, one can expect that the alpha -hydrogen derived from the substrate will be retained at the alpha -position of the product (25). Such an internal retention of the alpha -hydrogen was verified in the reactions catalyzed by two PLP-dependent racemases: amino acid racemase with low substrate specificity from P. striata (34) and alpha -amino-epsilon -caprolactam racemase from Achromobacter obae (35). Thus, it was supposed that these reactions proceed through a single-base mechanism (34, 35). However, Shostak and Schirch (36) argued in their studies on the mechanism of alanine racemization catalyzed by serine hydroxymethyltransferase that the internal retention of the substrate-derived alpha -hydrogen could also allow a two-base mechanism by assuming a hydrogen shuttle between the bases as proposed for the aconitase reaction; the latter enzyme is known to have a network of at least five interchangeable protons at the active site that only exchange slowly with the solvent (37). Although the reaction catalyzed by the amino acid racemase from P. striata was accompanied by clear internal retention of the alpha -hydrogen, a two-base mechanism has been proposed on the basis of the complete disagreement between the substrate enantiomers examined for the relative rates of deuterium incorporation from 2H2O into separate enantiomers (38). Our findings shown here indicate that the catalytic base(s) responsible for alpha -hydrogen abstraction and addition are situated on both faces of the plane of the substrate-cofactor complex. Our enzyme is assumed to be closely homologous to the P. striata enzyme, because the latter strain is now classified into the same group as P. putida. If this is the case, our enzyme would also use two bases for catalysis. Recently, Shaw et al. (39) clarified the three-dimensional structure of alanine racemase from Bacillus stearothermophilus. They suggested that Tyr-265 and Lys-39, the PLP binding lysine, serve as the bases. Sawada et al. have presented kinetic evidence to show that the alanine racemase reaction follows a two-base mechanism (40).

The stereospecificity for the hydrogen transfer reflects the structure of the active site of pyridoxal enzymes, especially the topographical relationship between the catalytic base for the hydrogen transfer and the bound coenzyme. Therefore, stereospecificity has been discussed in relation to the molecular evolution of the pyridoxal enzymes (17, 18, 24). No whole primary structure or three-dimensional structure of amino acid racemases with low substrate specificity has been determined. However, the primary structures of alanine racemases, which are homologous to the amino acid racemases in the sequences around the lysine residue binding PLP (41), show few similarities to those of other PLP enzymes (42). Therefore, amino acid racemase with low substrate specificity probably belongs to the same family of proteins as alanine racemase: a unique family containing only alanine racemase, mammalian ornithine decarboxylase, and meso-alpha ,epsilon -diaminopimelate (DAP) decarboxylase with little similarity to other PLP enzymes (43). We have shown that the decarboxylation of DAP catalyzed by DAP decarboxylase proceeds through inversion of the configuration of the alpha -carbon of DAP (29). This indicates that the enzyme function must be conducted on both sides of the plane of the substrate·PLP complex so as to decarboxylate on one side and to introduce a proton on the other side. The DAP decarboxylase reaction is homologous to the racemase reaction in this respect. It may be interesting to examine whether DAP decarboxylase and ornithine decarboxylase catalyze the removal of tritium nonstereospecifically from C-4' of both (4'S)- and (4'R)-[4'-3H]PMP in the same manner as amino acid racemase with low substrate specificity. Our preliminary results have shown that alanine racemase from B. stearothermophilus also catalyzes the nonstereospecific removal of tritium from both [4'-3H]PMPs. Whatever the results of the DAP decarboxylase and ornithine decarboxylase reactions may be, it is interesting to note the clear relationship between the stereospecificity of hydrogen transfer and families of PLP enzymes.

    FOOTNOTES

* This work was supported in part by the Research for the Future Program from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 81-774-38-3240; Fax: 81-774-38-3248; E-mail: esaki{at}scl.kyoto-u.ac.jp.

1 The abbreviations used are: PLP, pyridoxal 5'-phosphate; AspAT, aspartate aminotransferase; BCAT, branched chain L-amino acid aminotransferase; D-AAT, D-amino acid aminotransferase; PMP, pyridoxamine 5'-phosphate; Tricine, N-tris(hydroxymethyl)methylglycine; HPLC, high performance; DAP, meso-alpha ,epsilon -diaminopimelate.

2 Lim, Y.-H., Yoshimura, T., Kurokawa, Y., Esaki, N., and Soda, K, unpublished results.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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