Determinants of catalytic activity and stability of carbonic anhydrase II as revealed by random mutagenesis.

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