Chemical mapping of the active site of the glucoamylase from Aspergillus niger

A recently developed technique for the probing of the combining sites of lectins and antibodies, to establish the structure of the epitope that 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 kinetics of hydrolysis of both monodeoxygenation and mono-O-methylation at each of the seven hydroxyl groups in order to gain an estimate of the differential changes in the free energies of activation, Delta Delta G double dagger. As expected, from previous publications, both deoxygenation and O-methylation of OH-4 (reducing unit), OH-4', or OH-6' strongly 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 either OH-2' or OH-3' caused much smaller (0.96 and 0.52 kcal/mol) increases in Delta Delta G double dagger, the mono-O-methylations resulted in severe steric hindrance to the formation of the activated complex. The 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-isomaltoside was docked into the combining site of the X-ray crystal structure at 2.4 Angstrom resolution of the complex with the inhibitor acarbose. A fit free of steric interactions with the protein was found that has the methyl alpha-glucopyranoside unit in the normal C-4(1) conformation and the other glucose unit approaching a half-chair conformation with the interunit fragment defined by the torsion angles phi/psi/omega = 74 degrees/134 degrees/166 degrees (O-5'-C-1'O-psi-6(psi)-C-6(omega)-C-5-O-5). The model provides a network of hydrogen bonds that appears to well represent the activated complex formed by the glucoamylase with both maltose and isomaltose since the structures appear to provide a sound rationale for both the specificity and catalysis provided by the enzyme.