FUNCTIONAL-CHARACTERIZATION OF HUMAN CARBONIC-ANHYDRASE-II VARIANTS WITH ALTERED ZINC-BINDING SITES

Carbonic anhydrase II (CAII) contains a conserved Hiss zinc polyhedron which is essential for catalysis. Removal of any one of the His ligands by replacement with Ala decreases (approximate to 10(5)-fold), but does not abolish, zinc binding and increases the rate constant for zinc dissociation. CAII variants with a His ligand substituted with Cys, Asp, or Glu bind zinc only approximate to 10-fold better than a His(2) zinc polyhedron in CAII. The large decrease in zinc affinity (approximate to 5 kcal/mol) in these variants compared to the wild-type His(3) Site reflects mainly unfavorable compensatory protein structural rearrangements observed in the X-ray crystallographic structures of some of these CAII variants, described by Ippolito and Christianson (following paper in this issue). However, the zinc affinity of these sites is still higher than zinc polyhedra designed de novo. Substitution of the His zinc ligands with negatively charged amino acids both increases the pK(a) of the zinc-bound water by greater than or equal to 1.6 pH units, confirming that neutral ligands maintain the low zinc-water pK(a) and decreases the pH-independent k(cat)/K-M for ester hydrolysis (3-30-fold) and CO2 hydration (approximate to 10(3)-10(5)-fold). Additionally, decreases in the dissociation constant (approximate to 10(2)-10(5)-fold) for the transition state analog acetazolamide correlate with the decreased catalytic efficiency and increased pK(a) of these CAII variants. These data indicate that the histidine ligands, although not essential fdr catalysis, are conserved to maximize electrostatic stabilization of both the ground-state zinc-hydroxide and the negatively charged transition state. These studies provide valuable insights into the functional consequences of engineering a catalytic zinc site in a metalloenzyme.