Non-stereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity (MOLECULAR BIOFUNCTION-Molecular Microbial Science)

Abstract

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 non-stereospecifically 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 a-ketoglutarate through the transamination with L-ornithine. Because glutamate does not serve as a substrate for racemization, the enzyme catalyzed the non-stereospecific overall transamination between L-ornithine and a-ketoglutarate. The cleavage and formation of the C-H bond at C-4' of the coenzyme and C-2 of the substrate thus occurs non-stereospecifically 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 non-stereospecific transamination.