Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant

Abstract

To test the hypothesis that histidine 64 in the active site of human carbonic anhydrase II functions as a proton-transfer group in the catalysis of CO2 hydration, we have studied a site-specific mutant having histidine 64 replaced by alanine, which cannot transfer protons. The steady-state kinetics of CO2 hydration has been measured as well as the exchange of 18O between CO2 and water at chemical equilibrium. The results show that the rate of exchange between CO2 and HCO3- at chemical equilibrium is essentially unaffected by the amino acid substitution at pH greater than 7.0 and slightly decreased in the mutant at pH less than 7.0 (by a factor of 2 at pH 6.0). However, in the absence of buffer the rate of release from the active site of water bearing substrate oxygen is smaller by as much as 20-fold for the mutant as compared to unmodified enzyme. Furthermore, in the unmodified enzyme water release is inhibited by micromolar concentrations of Cu2+ ions, but no such inhibition is observed with the alanine 64 variant. These results suggest that the mutation has specifically affected the rate of proton transfer between the active site and the reaction medium. This kinetic defect in the mutant can be overcome by increasing the concentration of certain buffers, such as imidazole and 1-methylimidazole, but not by others buffers, such as MOPS or HEPES. Similarly, the maximal rate of CO2 hydration at steady state catalyzed by the alanine 64 variant is very low in the presence of MOPS or TAPS buffers but considerably higher in the presence of imidazole derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

Unexpected Binding Mode of the Sulfonamide Fluorophore 5-Dimethylamino-1-naphthalene Sulfonamide to Human Carbonic Anhydrase II

“…A conformational change is observed for His-64, the catalytic proton shuttle (Steiner et al, 1975;Tu et al, 1989); the imidazole side chain undergoes a rotation of 58°a bout side chain torsion angle 1 to the "out" conformation (data not shown), as observed in certain CAII structures (e.g. see Nair and Christianson (1991)).…”

Atomic Crystal and Molecular Dynamics Simulation Structures of Human Carbonic Anhydrase II: Insights into the Proton Transfer Mechanism,

“…The intramolecular proton transport occurs between the Zn-bound solvent and the side chain of His64, which is located on the edge of the active site, where the proton is further transferred out to the bulk solvent (eq 2). The mutation of His64 to an alanine (H64A HCA II) reduces the proton transfer activity 10-to 50-fold (7). Various crystal structures, determined at different pH values, have revealed that His64 occupies dual conformations, the socalled "in", pointing inward toward the active site, and "out", pointing outward away from the active site, and it is thought that the apparent flexibility of His64 is essential to its function as an efficient proton shuttle ( Figure 1) (8,9).…”

Positions of His‐64 and a bound water in human carbonic anhydrase II upon binding three structurally related inhibitors

“…experiments). Movement of the side chain of His-64 has also been implicated in the enzyme mechanism (Tu et al, 1989;Krebs et al, 1991;Nair & Christianson, 1991). Therefore, the observation of a change in the position of the side chain of His-64 between the native enzyme (Eriksson et al, 1988) and that inhibited by I C (Baldwin et al, 1992) (Fig.…”