COMPARISON OF INACTIVATION AND UNFOLDING OF GREEN CRAB (SCYLLA-SERRATA) ALKALINE-PHOSPHATASE DURING DENATURATION BY GUANIDINIUM CHLORIDE

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a met alloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enz yme during denaturation in guanidinium chloride (GuHCl) solutions of d ifferent concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity p reviously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 381-436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. Th e results show that inactivation occurs before noticeable conformation al change can be detected. It is suggested that the active site of gre en crab alkaline phosphatase containing multiple metal ions is also si tuated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.