Three mutants, Asp55-Gly, Tyr306-->Phe, and Asp309-->Asn, of Aspergillus awamori glucoamylase (identical to Aspergillus niger glucoamylase) were constructed to elucidate the roles of two conserved regions within fungal glucoamylases. Kinetic studies indicate that both of these regions are closely associated with activity. The Asp55-->Gly mutation decreases the kcat approximately 200 times toward maltose and isomaltose, while KM values remain similar to the wild-type. This localizes Asp55 to subsite 1 of glucoamylase where it affects catalytic activity, but not ground-state binding. The pKa value of the catalytic general acid, Glu179, is 1 pH unit lower in that mutant compared to wild-type enzyme, confirming the proximity of Asp55 to the site of catalysis. Tyr306-->Phe is highly active, but affects binding in subsite 2. It moreover shows enhanced binding in the fourth subsite, suggesting that the conserved region around residue 306 interacts with Trp120, a critical residue that directs conformational changes stabilizing the transition-state structure. Finally, the Asp309-->Asn mutation decreases the kcat for isomaltose hydrolysis around 200-fold, but only 30-fold for maltose. This specific effect on the hydrolysis of the alpha-1,6-linked substrate locates Asp309 to subsite 2. Substitution of Asp309 influences affinities of distant subsites, especially subsite 4, similar to mutations of other carboxylic acid residues situated near subsites 1 and 2.