Role reversal for substrates and inhibitors. Slow inactivation of D-amino acid transaminase by its normal substrates and protection by inhibitors.

摘要: D-Amino acid transaminase, which catalyzes the synthesis of D-alanine and D-glutamate for bacterial cell wall, is a candidate design specific inhibitors that could be novel antimicrobial agents. Under experimental conditions usually employed enzyme assays, kinetic parameters its substrates were determined short incubation periods, when intermediates products do not accumulate activity linear with time. Such analyses indicate accepts most D-amino acids but D-aspartate are best substrates. different type exposed to D-alanine, intermediates, periods hours, it slowly becomes inactivated (Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., Manning, J. (1992) Biochemistry 31, 6018-6023). We now report D-aspartate, D-glutamate, L-alanine also lead slow inactivation. Methylation or amidation alpha-COOH group prevents inactivation, indicating decarboxylation required inactivation; release CO2 from substrate demonstrated. The alpha-methyl analog showing alpha-hydrogen required, i.e. some processing required. Lys145, binds pyridoxal 5'-phosphate in wild-type enzyme, involved inactivation since two active site mutant enzymes, K145Q K145N, inactivated. Reactivation inactive at acidic pH accompanied by ammonia corresponding 1 mol/mol dimeric enzyme. Competitive inhibitors, amine-containing buffers, thiols effectively impede This reversal roles can an inactivator inhibitor act as protector, occurs during time period used measure steady-state kinetics initial velocities reactions have physiological relevance cells.