Subsite Structure ofRhizopusGlucoamylase for the Hydrolytic Reaction of Isomaltooligosaccharides

Abstract

The subsite structure of Rhizopus glucoamylase was identified for the hydrolytic reaction of isomaltooligosaccharides at 25°C and at pH 4.5. The arrangement of the subsite affinities Ai’s (i is the subsite number) was evaluated as follows: A1 = −6.0, A2 = 13.8, A3 = 4.6, A4 = 1.5, A5 = 0.6, A6= −0.5, and A7 = 0.2 (in kJ mol−1 unit). The intrinsic rate constant for the hydrolytic reaction in the productive binding mode of substrates, kint, was 10.2 s−1. Larger values of Km for isomaltooligosaccharides compared with those for maltooligosaccharides were attributed to smaller values of A1 and A2 compared with those for maltooligosaccharides, and smaller values of k0 were attributed to smaller values of A1 and kint.Steady-state inhibitory kinetic studies showed that both isomaltooligosaccharides and maltooligosaccharides bind to the same active site of the enzyme.