Conformational Mobility of His-64 in the Thr-200 → Ser Mutant of Human Carbonic Anhydrase II

Abstract

The three-dimensional structure of the Thr-200 → Ser (T200S) mutant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods at 2.1-Å resolution. This particular mutant of CAII exhibits CO2 hydrase activity that is comparable to that of the wild-type enzyme with a 2-fold stabilization of the E·HCO3− complex and esterase activity that is 4-fold greater than that of the wild-type enzyme. The structure of the mutant enzyme reveals no significant local changes accompanying the conservative T200S substitution, but an important nonlocal structural change is evident: the side chain of catalytic residue His-64 rotates away from the active site by 105° about χ1 and apparently displaces a water molecule. The displaced water molecule is present in the wild-type enzyme; however, the electron density into which this water is built is interpretable as an alternate conformation of His-64 with 10-20% occupancy. The rate constants for proton transfer from the zinc-water ligand to His-64 and from His-64 to bulk solvent are maintained in the T200S variant; therefore, if His-64 is conformationally mobile about χ1 and/or χ2 during catalysis, compensatory changes in solvent configuration must sustain efficient proton transfer. © 1991, American Chemical Society. All rights reserved.