KINETICS OF INHIBITION OF AMINOACYLASE ACTIVITY BY DITHIOTHREITOL OR 2-MERCAPTOETHANOL

The previously described kinetic method of the substrate reaction duri ng irreversible inhibition of enzyme activity [Tsou (1988) Adv. Enzymo l. Relat. Areas Mol. Biol. 61, 381-436] has been used to study the ina ctivation kinetics of aminoacylase by dithiothreitol (DTT) and 2-merca ptoethanol (MET). The results show that the inactivation of aminoacyla se by DTT or MET is competitive slow-reversible inhibition. The micros copic rate constants for the inactivation reaction were determined. Re moval of these inhibitors by dialysis can lead to complete recovery of enzymatic activity. The present results also show that the presence o f equimolar Zn2+ to DTT gives complete protection of the enzyme agains t the inactivation by DTT. Moreover, addition of equimolar amounts of Zn2+ to DTT can induce recovery of the enzymatic activity of DTT-inact ivated enzyme. It is known that aminoacylase from pig kidney contains no disulfide bonds. Therefore, it may be suggested that inactivation o f aminoacylase by dithiothreitol or 2-mercaptoethanol is not due to th e reduction of disulfide bonds, and is a competitive slow-reversible i nhibition. (C) Munksgaard 1996.