STRUCTURE AND ENERGETICS OF THE GLUCOAMYLASE-ISOMALTOSE TRANSITION-STATE COMPLEX PROBED BY USING MODELING AND DEOXYGENATED SUBSTRATES COUPLED WITH SITE-DIRECTED MUTAGENESIS

Molecular recognition, site-directed mutagenesis, and molecular modeli ng are combined to describe hydrogen bonds important for formation and catalysis of the Aspergillus niger glucoamylase-isomaltose complex. T his analysis of the energetics of the transition-state complex identif ied OH-4', -6', and -4 as critical for isomaltose hydrolysis. Side-cha ins hydrogen bonded to isomaltose OH-4 (reducing end unit, i.e. at glu coamylase binding subsite 2) induced substrate conformation adjustment to optimize binding energy contributed by charged hydrogen bonds to O H-4' and -6' at the non-reducing unit (i.e. at subsite 1). These inter actions were evident in the modeled complex of glucoamylase and isomal tose in the preferred trans-gauche conformation. Kinetic analysis demo nstrated reductions in k(cat) of 10(3) to 10(5)-fold for the correspon ding deoxy- and O-methyl analogs of isomaltose. Analysis of two mutant s at the level of subsite 2, Glu180 --> Gln and Asp309 --> Glu, showed the binding energy for the enzyme-transition state complex, Delta Del ta G double dagger, contributed by OH-3 and -4 to be 6-7 kJ mol(-1) we aker than with wild-type enzyme. Unexpectedly, however, substitution o f isomaltose OH-4' and -6' (at subsite 1) resulted in 10 to 12 kJ mol( -1) lower Delta Delta G double dagger for both the mutants. Mutation a t subsite 2, therefore, strongly perturbed distant transition-state st abilizing interactions. This was confirmed with 4'- and 6'-deoxy analo gs of the conformationally biased methyl 6-R-C-methyl-alpha-isomaltosi de, readily adopting trans-gauche conformation, that exhibit full Delt a Delta G double dagger of 18 to 20 kJ mol(-1) for both mutants and wi ld-type. Glucoamylase, during catalysis, thus seems to induce a change from the predominant solution gauche-gauche conformer to tuans-gauche isomaltose. This leads to enhanced binding at subsite 1 in the enzyme transition-state complex. (C) 1996 Academic Press Limited