STRUCTURAL AND FUNCTIONAL IMPORTANCE OF A CONSERVED HYDROGEN-BOND NETWORK IN HUMAN CARBONIC ANHYDRASE-II

Amino acid substitutions at Thr199 of human carbonic anhydrase II (CAI I) (Thr199 --> Ser, Ala, Val, and Pro) were characterized to investiga te the importance of a conserved hydrogen bonding network. The three-d imensional structures of azide-bound and sulfate-bound T199V CAIIs wer e determined by x-ray crystallographic methods at 2.25 and 2.4 angstro m, respectively (final crystallographic R factors are 0.173 and 0.174, respectively). The CO2 hydrase activities of T199S and T199P variants suggest that the side chain methyl and backbone amino functionalities stabilize the transition state by almost-equal-to 0.4 and 0.8 kcal/mo l, respectively. The side chain hydroxyl group causes: stabilization o f zinc-hydroxide relative to zinc-water (pK(a) increases almost-equal- to 2 units); stabilization of the transition state for bicarbonate deh ydration relative to the CAII.HCO3- complex (almost-equal-to 5 kcal/mo l); and destabilization of the CAII.HCO3- complex (almost-equal-to 0.8 kcal/mol). An inverse correlation between log(k(cat)CO2/K(M)) and the pk(a) of zinc-water (r = 0.95, slope = -1) indicates that the hydroge n bonding network stabilizes the chemical transition state and zinc-hy droxide similarly. These data are consistent with the hydroxyl group o f Thr199 forming a hydrogen bond with the transition state and a non-h ydrogen-bonded van der Waals contact with CAII.HCO3-.