The Catalytic Mechanism of Carbonic Anhydrase

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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 + ⇌ 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 ⇌ 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.