Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Abstract

Glucoamylases G1 and G2 from Aspergillus niger lost the catalytic activity by prolonged treatment with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide(EAC). A total of 23 and 16 carboxyl groups were substituted out of 71 and 54 present in the G1 and G2 enzyme, respectively. The kinetics and the pH-dependence of the inactivation were compatible with the existence of one essential carboxyl group with a pKa 5.5. The inhibitor acarbose (a pseudotetrasaccharide) and the substrate maltose prevented EAC-modification of 4 and 2 carboxyl groups, respectively, with considerable retention of enzyme activity. Following removal of these ligands, differential labelling of critical carboxyl residue(s) was achieved by means of [3H]EAC. Glucoamylase G2 with 12 EAC groups incorporated, prepared in the presence of acarbose, displayed a two-fold increase of Km and a slight reduction of Vmax for hydrolysis of starch relative to the unmodified enzyme. Both Km and Vmax for hydrolysis of maltose were almost unaffected. In addition, maltose and three inhibitors perturbed the UV absorbance spectrum of this derivative. An altered shape of the acarbose-induced difference spectrum implied substitution of a carboxyl group near a binding tryptophanyl residue at a distance from the catalytic site. Efficient protection by acarbose against only the second part of the biphasic course of inactivation similarly reflected that residues playing different roles in the mechanism of action reacted with EAC. In separate experiments, evidence was obtained for a preferred reaction of ethylenimine with enzymatically important carboxyl groups in glucoamylase.