Subsite Structure of Rhizopus Glucoamylase 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 A_i'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, k_int, was 10.2s^-1. Larger values of K_m 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 k_int. Steady-state inhibitory kinetic studies showed that both isomaltooligosaccharides and maltooligosaccharides bind to the same active site of the enzyme.