A program of cassette mutagenesis was undertaken in the active site of Aspergillus awamori glucoamylase, mainly to alter the effect of pH dependence of the enzyme but also to determine possible functions of the mutated residues. For this purpose, nine single amino acid mutants were prepared and expressed in Saccharomyces cerevisiae, and their effects were determined by using steady-state enzyme kinetics;Three different groups of mutations were designed. In the first group, Glu179 → Asp, Asp176 → Glu, and Glu180 → Asp mutations were prepared. The Glu179 → Asp mutation led to a very large decrease in k[subscript] cat but no change in K[subscript] M, verifying the catalytic function of this residue. The effects of Asp176 → Glu and the Glu180 → Asp mutations were more pronounced for K[subscript] M than k[subscript] cat, implying a possible function for Asp176 and Glu180 in substrate binding or structural integrity;The second category comprised the Leu177 → Asp, Trp178 → Asp, Val181 → Asp, and Asn182 → Asp mutations. The Trp178 → Asp mutation caused a very large decrease in the k[subscript] cat value and an increase in the K[subscript] M value, showing the importance of Trp178 in the active site. The Leu177 → Asp mutation decreased k[subscript] cat value only moderately, probably by changing the position of the general acid catalytic group. The last two mutations in this group, Val181 → Asp and Asn182 → Asp, caused slight changes in kinetic values, suggesting that Val181 and Asn182 were of minor importance both catalytically and structurally;Finally, the third category contained two insertion mutations, where Asp and Gly were added between residues 176 and 177. These two mutations were prepared based on the homology between glucoamylases and [alpha]-amylases, [alpha]-glucosidases, and transglucosylases. However, the mutations resulted in an almost complete loss of activity, probably caused by destruction of the active site structure;The effects of Glu179 → Asp, Trp178 → Asp, and the insertion mutations on the pH dependence of glucoamylase could not be determined due to very low or undetectable activities, while the others did not affect this property significantly.