The catalytic mechanism of carbonic anhydrase

Abstract

It is shown that an "inverse" relationship between the pH dependencies of the rates of hydration of CO(2) and dehydration of HCO(3) (-) by carbonic anhydrase (EC 4.2.1.1) is a direct consequence of the thermodynamic equilibrium between CO(2) and HCO(3) (-) and independent of any assumptions about the catalytic mechanism. It is further shown that proposed mechanisms for carbonic anhydrase involving HCO(3) (-) as the substrate in the dehydration reaction and a proton transfer reaction, EH(+) right harpoon over left harpoon E + H(+), as an obligatory step during catalysis obey the rule of microscopic reversibility. This includes mechanisms in which the proton dissociation is from a zinc-coordinated water molecule. Such mechanisms can be in accord with the observed rapid turnover rates of the enzyme, since rapid proton exchange can occur with the buffer components, EH(+) + B right harpoon over left harpoon E + BH(+). Mechanisms in which H(2)CO(3) is the substrate in dehydration avoid the proton-transfer step, but require that H(2)CO(3) combines with enzyme more rapidly than in a diffusion-controlled reaction. Physico-chemical evidence for and against a zinc-hydroxide mechanism is discussed.