B. Elleby et al., Enhancement of catalytic efficiency by the combination of site-specific mutations in a carbonic anhydrase-related protein, EUR J BIOCH, 267(19), 2000, pp. 5908-5915
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.