CHEMICAL MAPPING OF THE ACTIVE-SITE OF THE GLUCOAMYLASE OF ASPERGILLUS-NIGER

A recently developed technique for the probing of the combining sites of lectins and antibodies, to establish the structure of the epitope t hat is involved in the binding of an oligosaccharide, is used to study the binding of methyl alpha-isomaltoside by the enzyme glucoamylase. The procedure involved the determination of the effects on the kinetic s of hydrolysis of both monodeoxygenation and mono-O-methylation at ea ch of the seven hydroxyl groups in order to gain an estimate of the di fferential changes in the free energies of activation, Delta Delta G d ouble dagger. As expected, from previous publications, both deoxygenat ion and O-methylation of OH-4 (reducing unit), OH-4', or OH-6' strongl y hindered hydrolysis, whereas the kinetics were virtually unaffected by either the substitutions at OH-2 or structural changes at C-1. The substitutions at OH-3 caused increases of 2.1 and 1.9 kcal/mol in the Delta Delta G double dagger. In contrast, whereas deoxygenation of eit her OH-2' or OH-3' caused much smaller (0.96 and 0.52 kcal/mol) increa ses in Delta Delta G double dagger, the mono-O-methylations resulted i n severe steric hindrance to the formation of the activated complex. T he relatively weak effects of deoxygenation suggest that the hydroxyl groups are replaced by water molecules and thereby participate in the binding by contributing effective complementarity. Methyl alpha-isomal toside was docked into the combining site of the X-ray crystal structu re at 2.4 Angstrom resolution of the complex with the inhibitor acarbo se. A fit free of steric interactions with the protein was found that has the methyl alpha-glucopyranoside unit in the normal C-4(1) conform ation and the other glucose unit approaching a half-chair conformation with the interunit fragment defined by the torsion angles phi/psi/ome ga = 74 degrees/134 degrees/166 degrees (O-5'-C-1'O-psi-6(psi)-C-6(ome ga)-C-5-O-5). The model provides a network of hydrogen bonds that appe ars to well represent the activated complex formed by the glucoamylase with both maltose and isomaltose since the structures appear to provi de a sound rationale for both the specificity and catalysis provided b y the enzyme.