Fungal glucoamylases contain four conserved regions. One region from the Aspergillus niger enzyme contains three key carboxylic acid residues, the general acid catalytic group, Glu179, along with Asp176 and Glu180. Three site-directed mutations, Leu177-->His, Trp178-->Arg and Asn182-->Ala, were constructed near these acidic groups to reveal the function of other conserved residues in this region. Leu177 and Trp178 are strictly conserved among fungal glucoamylases, while an amide, predominantly Asn, always occurs at position 182. Substitutions of Leu177 or Trp178 cause significant decreases in kcat with the substrates tested. Similar increases in activation energies obtained with Leu177-->His with both alpha-(1,4)- and alpha-(1,6)-linked substrates indicate Leu177 is located in subsite 1. KM values obtained with the Trp178-->Arg mutation increase for an alpha-(1,6)-linked substrate, but not for alpha-(1,4)-linked substrates. Calculated differences in activation energy between substrates indicate Trp178 interacts specifically with subsite 2. The Asn182-->Ala mutation did not change kcat or KM values, indicating that Asn182 is not crucial for activity. These results support a mechanism for glucoamylase catalytic activity consisting of a fast substrate binding step followed by a conformational change at subsite 1 to stabilize the transition state complex.