Inhibition kinetics of green crab (Scylla serrata) alkaline phosphatase activity by dithiothreitol or 2-mercaptoethanol

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloe nzyme which catalyzes the nonspecific hydrolysis of phosphate monoesters. S ome pollutants in seawater affect the enzyme activity causing loss of the b iological function of the enzyme, which affects the exuviating crab-shell a nd threatens the survival of the animal. The present paper studies the effe cts of thiohydroxyal compounds on the activity of green crab alkaline phosp hatase. The results show that thiohydroxyal compounds can lead to reversibl e inhibition. The equilibrium constants have been determined for dithiothre itol (DTT) and mercaptoethanol (ME) binding with the enzyme and/ or the enz yme-substrate complexes. The results show that both DTT and ME are non-comp etitive inhibitors. The kinetics of enzyme inactivation by ME at low concen trations has been studied using the kinetic method of the substrate reactio n. The results suggest that at pH 10.0, the action of ME on green crab ALP is first quick equilibrium binding and then slow inactivation. The microsco pic rate constants were determined for inactivation and reactivation. The r ate constant of the forward inactivation (k(+0)) is much larger than that o f the reverse reactivation (k(-0)). Therefore, when the ME concentration is sufficiently large, the enzyme is completely inactivated. (C) 2000 Elsevie r Science Ltd. All rights reserved.