Enhancement of catalytic efficiency by the combination of site-specific mutations in a carbonic anhydrase-related protein

A single mutation, involving the replacement of an arginine residue with hi stidine to reconstruct a zinc-binding site, suffices to change a catalytica lly inactive murine carbonic anhydrase-related protein (CARP) to an active carbonic anhydrase with a CO2-hydration turnover number of 1.2 x 10(4) s(-1 ). Further mutations, leading to a more 'carbonic anhydrase-like' active-si te cavity, results in increased activity. A quintuple mutant having His94, Gln92, Val121, Val143, and Thr200 (human carbonic anhydrase I numbering sys tem) shows k(cat) = 4 x 10(4) s(-1) and k(cat)/K-m = 2 x 10(7) M-1.s(-1), g reatly exceeding the corresponding values for carbonic anhydrase isozyme II I and approaching those characterizing carbonic anhydrase I. In addition, a buffer change from 50 mM Taps/NaOH to 50 mM 1,2-dimethylimidazale/H2SO4 at pH 9 results in a I l-fold increase in k(cat) for this quintuple mutant. T he CO2-hydrating activity of a double mutant with His94 and Gln92 shows com plex pi-I-dependence, but the ether mutants investigated behave as if the a ctivity (k(cat)/K-m) is controlled by the basic form of a single group with pK(a) near 7.7. In a similar way to human carbonic anhydrase II, the buffe r behaves formally as a second substrate in a ping-pong pattern, suggesting that proton transfer between a zinc-bound water molecule and buffer limits the maximal rate of catalysis in both systems at low buffer concentrations . However, the results of isotope-exchange kinetic studies suggest that pro ton shuttling via His64 is insignificant in the CARP mutant in contrast wit h carbonic anhydrase II. The replacement of ne residues with Val in positio ns 121 or 143 results in measurable 4-nitrophenyl acetate hydrolase activit y. The pH-rate profile for this activity has a similar shape to those of ca rbonic anhydrase I and II. CD spectra of the double mutant with His94 and G ln92 are variable, indicating an equilibrium between a compact form of the protein and a 'molten globule'-like form. The introduction of Thr200 seems to stabilize the protein.