A simple method for determination of stereospecificity of aminotransferases for C-4' hydrogen transfer of the coenzyme.
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 3H2O 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, alpha-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.
Department of Applied Chemistry and Biotechnology, Yamanashi University, Japan.
K
K
Nishimura
J
J
Ito
T
T
Yoshimura
N
N
Esaki
K
K
Soda
D000410:Alanine Transaminase / Q000378:metabolism*; D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology; D051263:D-Alanine Transaminase; D004926:Escherichia coli / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011730:Pyridoxal / Q000378:metabolism*; D011733:Pyridoxamine / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013552:Swine; D000637:Transaminases / Q000378:metabolism*
D001051:Apoenzymes; D011730:Pyridoxal; D011733:Pyridoxamine; D000637:Transaminases; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase