SITE-DIRECTED MUTAGENESIS OF THE CATALYTIC BASE GLUTAMIC-ACID-400 IN GLUCOAMYLASE FROM ASPERGILLUS-NIGER AND OF TYROSINE-48 AND GLUTAMINE-401, BOTH HYDROGEN-BONDED TO THE GAMMA-CARBOXYLATE GROUP OF GLUTAMIC-ACID-400

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 en zyme, K-m values for maltose and maltoheptaose are 12- and 3-fold high er for the Glu400-->Gln mutant, with k(cat) values 35- and 60-fold low er, respectively, for the same substrates. This unusually high residua l activity for a glycosylase mutant at a putative catalytic group is t entatively explained by a reorganization of the hydrogen bond network, using the crystal structure of the related Aspergillus awamori var. X 100 glucoamylase in complex with 1-deoxynojirimycin [Harris, E. M. S., Aleshin, A. E., Firsov, L. M., and Honzatko, R. B. (1993) Biochemistr y 32, 1618-1626]. Supposedly Gln400 in the mutant hydrogen bonds to th e invariant Tyr48, as does Glu400 in the wild-type enzyme. For Tyr48-- >Trp A. niger glucoamylase k(cat) is reduced 80-100-fold, while K-m is increased only 2-3-fold. Gln401 also hydrogen bonds to Glu400, but it s mutation to glutamic acid has only a minor effect on activity. The T yr48-->Trp and Glu400-->Gln glucoamylases share particular features in displaying unusually high activity below pH 4.0-which reflects lack o f the wild-type catalytic base function-and unusually low binding affi nity at subsite 2. Both mutants have lost 13-16 kJ mol(-1) in transiti on-state stabilization energy. The Glu400-->Gln mutant confirms the ro le of Glu400 in catalysis, and mutation of Tyr48 suggests that this si de chain is functionally Linked to Glu400 and is important for maintai ning the active site geometry and for stabilization of an oxocarbonium ion substrate intermediate. The properties of the glucoamylase mutant s are compared with results of mutational analysis in other carbohydra ses.