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 (4S)-or (4R)-[4-3 H]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 ␣-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 ␣-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.Although enzymatic racemization of amino acid is apparently simple, consisting of a nonstereospecific rearrangement of the substrate ␣-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 ␣-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 5Ј-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 ␣-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 regener...