INSERTION OF A BULKY RESIDUE ADJACENT TO THE PROTON SHUTTLE GROUP IN CAII SIGNIFICANTLY DECREASES THE EFFICIENCY OF PROTON TRANSFER. Laura Scolnick^*, Jane E. Jackman^#, Kenneth M. Merz⁺, Jr., David W. Christianson^*, Carol A. Fierke^{# *}Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323^{, #}Department of Biochemistry, Duke University Medical Center, Box 371, Durham, North Carolina 27710, ⁺Chemistry Department, Pennsylvania State University, University Park, PA 16802

Human carbonic anhydrase II (CAII) is a zinc metalloenzyme that catalyzes the reversible hydration of CO₂ to HCO₃^- and a proton in a two-step mechanim. First, zinc-bound hydroxyl attacks the carbonyl carbon of CO₂ to form a zinc-bound bicarbonate that is subsequently replaced by water to release product bicarbonate. The second step, which regenerates the active site zinc-hydroxide species, involves the transfer of a proton from zinc-bound water to a solvent buffer molecule,this is the rate-limiting step for CO₂ hydration at high substrate concentrations. Kinetic data and high resolution x-ray crystallographic structures of E. coli expressed CAII variants A65F, A65H, A65L, A65T, A65S, and A65G reveal that larger amino acids substituted at position 65 protrude into the active site and block proton transfer between the active site zinc bound water and histidine 64. In addition, evolutionary drift of position 65 among the CA isozymes shows that position 65 has evolved to be a small amino acid.