The functional importance of a conserved hydrophobic face in human carbonic anhydrase II (CAII), including amino acid residues 190-210, was investigated by random mutagenesis. The catalytic activity, inhibitor binding, and level of CAII expression in Escherichia coli of 57 single amino acid variants were measured revealing that the function of amino acids correlates with their secondary structure placement. Side chains of amino acids in beta-sheet structure are required for the formation of folded, stable protein while those in the turn region determine catalytic efficiency and inhibitor specificity. The CAII active site is extremely plastic, accommodating amino acid substitutions of varied size, charge, and hydrophobicity with little effect on catalysis; only substitutions at Leu198 and Thr199 decrease the rates of CO2 hydration and ester hydrolysis more than 5-fold. These results pinpoint the hydrogen bond network, including the zinc-solvent molecule and Thr199, as crucial for high catalytic efficiency and also suggest that Leu198 forms a portion of a CO2 association site. Increased activity is observed for substitutions at Thr200 (esterase) and Leu203 (hydrase). In addition, the pKa of the zinc-bound water molecule varies upon substitution of amino acids which alter the overall charge of the active site. Three residues interact with sulfonamide inhibitors; substitutions at Thr199 decrease binding (up to 10(3)-fold) while mutations at Thr200 and Cys206 increase binding of dansylamide (up to 80-fold). Mutations in the beta-sheet structure (Asp190-Ser197 and Val207-Ile-210) decrease the protein expression of CAII in E. coli, causing the formation of insoluble protein aggregates in many cases. This may suggest an important role for these residues in the folding process. In addition, mutations in Trp192, cis-Pro202, and Trp209 increase thermal lability (up to 5000-fold).