A comparison of the kinetic properties of native bovine muscle carbonic anhydrase and an activated derivative with modified thiol groups

Abstract

Steady-state and equilibrium kinetic properties of native bovine carbonic anhydrase III (carbonate hydrolyase, EC 4.2.1.1) and a derivative modified with methyl methanethiosulfonate were investigated. The modified enzyme has a markedly increased CO 2 hydration activity compared to the native form with a 3-times higher value of k cat and a 6–10-times higher value of k cat K m . Qualitatively, the activated enzyme shows the same kinetic behavior as native isoenzyme III. This is reflected in similar pH dependences of the kinetic parameters for CO 2 hydration, similar solvent hydrogen isotope effects on these parameters, similar deviations from Michaelis-Menten kinetics for the HCO 3 − dehydration reaction, and similar behavior of the kinetics of CO 2 HCO 3 − exchange at chemical equilibrium as measured by a 13C-NMR magnetization transfer technique. It is concluded that the conversion of -SH groups to -S-S-CH 3 moieties does not change the catalytic mechanism, but leads to an increased rate of CO 2 HCO 3 − interconversion as well as to an increased rate of proton transfer between the active site and the reaction medium.