Reversal of the hydrogen bond to zinc ligand histidine-119 dramatically diminishes catalysis and enhances metal equilibration kinetics in carbonic anhydrase II

Direct metal ligands to transition metals in metalloproteins exert a profound effect on protein-metal affinity and function, Indirect ligands, i.e., second-shell residues that hydrogen bond to direct metal ligands, typically exert more subtle effects on the chemical properties of the protein-metal complex. However, E117 of human carbonic anhydrase II (CAII), which is part of the E117-119-Zn2+ triad, is a notable exception: E117-substituted CAIIs exhibit dramatically increased kinetics of zinc complexation, and the E117Q variant exhibits enormously diminished catalytic activity and sulfonamide affinity, The three-dimensional structures of zinc-bound and zinc-free El 17Q CAII reveal no discrete structural changes in the active site that are responsible for enhanced zinc equilibration kinetics and decreased activity, Additionally: the structure of the acetazolamide complex is essentially identical to that of the wild-type enzyme despite the 10(4)-fold loss of enzyme-inhibitor affinity. We conclude, therefore, that the functional differences between E117Q and wild-type CAIIs arise from electrostatic and not structural differences in the active site, We propose that the E117Q substitution reverses the polarity of the residue 117-H119 hydrogen bond, thereby stabilizing H119 as a histidinate anion in the E117Q CAII holoenzyme. The additional negative charge in the first coordination sphere of the metal ion increases the pK(a) of the zinc-water ligand, destabilizes the transition state for CO2 hydration, and facilitates the exchange of a zinc-histidine ligand with an additional water molecule by decreasing the stability of the tetrahedral zinc complex, These novel properties engineered into E117Q CAII facilitate the exploitation of CAII as a rapid and sensitive Zn2+ biosensor.