Replacement of the catalytic base Glu400 by glutamine in glucoamylase from Aspergillus niger affects both substrate ground-state binding and transition-state stabilization. Compared to those of the wild-type enzyme, Km values for maltose and maltoheptaose are 12- and 3-fold higher for the Glu400→Gln mutant, with kcat values 35- and 60-fold lower, respectively, for the same substrates. This unusually high residual activity for a glycosylase mutant at a putative catalytic group is tentatively explained by a reorganization of the hydrogen bond network, using the crystal structure of the related Aspergillus awamori var. X100 glucoamylase in complex with 1-deoxynojirimycin [Harris, E. M. S., Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1993) Biochemistry 32, 1618‒1626]. Supposedly Gln400 in the mutant hydrogen bonds to the invariant Tyr48, as does Glu400 in the wild-type enzyme. For Tyr48→Trp A. niger glucoamylase Kcat is reduced 80‒100-fold, while Km is increased only 2‒3-fold. Gln401 also hydrogen bonds to Glu400, but its mutation to glutamic acid has only a minor effect on activity. The Tyr48→Trp and Glu400→Gln glucoamylases share particular features in displaying unusually high activity below pH 4.0‒which reflects lack of the wild-type catalytic base function‒and unusually low binding affinity at subsite 2. Both mutants have lost 13‒16 kJ mol−1 in transition-state stabilization energy. The Glu400→Gln mutant confirms the role of Glu400 in catalysis, and mutation of Tyr48 suggests that this side chain is functionally linked to Glu400 and is important for maintaining the active site geometry and for stabilization of an oxocarbonium ion substrate intermediate. The properties of the glucoamylase mutants are compared with results of mutational analysis in other carbohydrases.
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