A CATALYTIC AMINO-ACID AND PRIMARY STRUCTURE OF ACTIVE-SITE IN ASPERGILLUS-NIGER ALPHA-GLUCOSIDASE

The catalytic amino acid residue of Aspergillus niger alpha-glucosidas e (ANGase) was identified by modification with conduritol B epoxide (C BE), a mechanism-based irreversible inactivator, The inactivation by C BE followed pseudo-first order kinetics, The interaction of CBE and AN Gase conformed to a model with a reversible enzyme-inhibitor complex f ormed before covalent inactivation, A competitive inhibitor, Tris, dec reased the inactivation rate, The incorporation of one mole of CBE per mole of ANGase was completely abolished the enzyme activity, A dissoc iated carboxyl group (-COO-) in the active site was suggested to attac k the C-1 of CBE, ANGase was composed of two subunits (P1 and P2), of which P2 was modified by CBE. The labelled residue was included in a p eptide (LY3) that was obtained from Lys-C protease digestion of CBE-bo und P2. The sequence analysis of CBE-labelled LY3 showed that an Asp w as the modified residue, that is, one of the catalytic amino acid resi dues of ANGase, The primary structure of LY3 was determined by analyzi ng the sequence of peptide fragments prepared by several proteases.