George C. Wynns

Role of Buffer in Catalysis of the Hydration of CO2 by Carbonic Anhydrase

Measurements of the rates of 18O exchange processes catalyzed by carbonic anhydrase have been a useful complement to the study by other kinetic techniques of the catalytic mechanism of CO2 hydration. It was the observation of 18O exchange between CO2 and water that first confirmed the participation of buffer in the catalysis (Silverman and Tu, 1975) and gave an estimate of the residence time of oxygen at the active site (Silverman and Tu, 1976). In this report 18O depletion from CO2 catalyzed by carbonic anhydrase is used to describe the rate of exchange of water at the enzyme’s active site and to elucidate further the role of buffer in the catalysis of CO2 hydration.

The Catalytic Mechanism of Carbonic Anhydrase Studied by 18O Exchange

Measurement by mass spectrometry of the exchange of 18O between CO2 and H2O at chemical equilibrium has yielded the rates of two steps in the hydration of CO2 catalyzed by carbonic anhydrase II. First, the rate of interconversion of CO2 and \(HCO_3^ - \), R1, occurs without rate-limiting proton transfer. Second, the rate of release from the enzyme of water bearing substrate oxygen RH20 occurs with rate-limiting proton transfer. The effects of three types of inhibitors on these rates is reported. Anions inhibit R1 and RH20 with the same inhibition constant by binding to the enzyme’s active site. Cu2+ and Hg2+ inhibit RH20 with KI near 1 x 10−7 M apparently by blocking the proton transfer step; these metal ions have negligible effects on R1. Phenol inhibits R1 with KI of 16 mM at pH 7.4 and enhances RH20, probably by binding to 18O-labeled enzyme.