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

Abstract

A single mutation, involving the replacement of an arginine residue with histidine to reconstruct a zinc-binding site, suffices to change a catalytically inactive murine carbonic anhydrase-related protein (CARP) to an active carbonic anhydrase with a CO2-hydration turnover number of 1.2 x 104 s-1. Further mutations, leading to a more 'carbonic anhydrase-like' active-site cavity, results in increased activity. A quintuple mutant having His94, Gln92, Val121, Val143, and Thr200 (human carbonic anhydrase I numbering system) shows kcat = 4 x 104 s-1 and kcat/Km = 2 x 107 M-1.s-1, greatly exceeding the corresponding values for carbonic anhydrase isozyme III and approaching those characterizing carbonic anhydrase I. In addition, a buffer change from 50 mM Taps/NaOH to 50 mM 1, 2-dimethylimidazole/H2SO4 at pH 9 results in a 14-fold increase in kcat for this quintuple mutant. The CO2-hydrating activity of a double mutant with His94 and Gln92 shows complex pH-dependence, but the other mutants investigated behave as if the activity (kcat/Km) is controlled by the basic form of a single group with pKa near 7.7. In a similar way to human carbonic anhydrase II, the buffer 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 proton shuttling via His64 is insignificant in the CARP mutant in contrast with carbonic anhydrase II. The replacement of Ile residues with Val in positions 121 or 143 results in measurable 4-nitrophenyl acetate hydrolase activity. The pH-rate profile for this activity has a similar shape to those of carbonic anhydrase I and II. CD spectra of the double mutant with His94 and Gln92 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.