35] Carbonic anhydrase: Oxygen-18 exchange catalyzed by an enzyme with rate-contributing Proton-transfer steps

Abstract

This chapter reviews the application of an 18 O exchange method to study the catalysis of the hydration of CO 2 by carbonic anhydrase, which, with a maximal turnover number near 10 6 sec –1 , is one of the fastest known enzymic reactions. The catalysis is so rapid that proton transfer steps between the enzyme and its environment, which are not ordinarily rate limiting for slower enzymic reactions, become rate-limiting and more amenable to investigation with carbonic anhydrase. The 18 O exchange method is chosen to investigate this problem because it is an equilibrium method that may be used in the absence of buffer or in the presence of excess buffer concentration, and buffers are sources of protons for transfer to the enzyme. With this method, it is possible to elucidate the role of proton transfer in the pathway of the catalytic hydration of CO 2 . A further result is that the fate of oxygen in the catalysis can be followed; hence, this method reveals the rate of release from the enzyme of water bearing substrate oxygen, when the substrate is 18 O-labeled bicarbonate.