Abstract
A simple method was established for determination of the stereospecificity of C-4′ hydrogen transfer of the coenzymes (pyridoxal and pyridoxamine). The method is based on the findings that aspartate aminotransferase of pig heart and D-amino acid aminotransferase of Bacillus sp. YM-1 catalyze the abstraction of the pro-S and pro-R proton at C-4′ of pyridoxamine, respectively. Pyridoxal is a poor coenzyme, but readily released from the enzyme. It reacts in 3H 2O with a substrate amino acid and an apo-aminotransferase whose stereospecificity for C-4′ hydrogen transfer is to be determined. The resultant pyridoxamine which is tritiated at C-4′ is incubated with an apo form of aspartate aminotransferase or D-amino acid aminotransferase and a substrate, α-keto acid. The stereospecificity for the C-4′ hydrogen transfer examined is determined by measurement of radioactivity retained in the pyridoxal formed. We showed by means of this method that C-4′ hydrogen transfer of coenzyme occurs on the si face of the external Schiff base in the transamination reactions of two aspartate aminotransferases of Bacillus sp. YM-2 and Escherichia coli, and aromatic amino acid aminotransferase of E. coli.
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