18] Proton transfer in carbonic anhydrase measured by equilibrium isotope exchange

Abstract

Kinetic and structural studies demonstrate the pathways of inter- and intramolecular proton transfer in the hydration of CO2 catalyzed by carbonic anhydrase. A histidine residue at position 64 is the most efficient of the carbonic anhydrases, carbonic anhydrase II, and acts as a transfer group shuttling protons between the buffer in solution and the zinc-bound water at the active site. This conclusion is confirmed by mutations to replace His-64 that results in the inactivation of catalysis. Mutations to introduce His-64 in the less efficient carbonic anhydrase III results in activation, with the mutant showing proton transfer properties, similar to the efficient form of the enzyme. In a series of site-specific mutants of isozyme III, the rate constants for proton transfer obeys a Brønsted correlation and show sharp curvature characteristic of facile proton transfers. Application of Marcus rate theory shows that this proton transfer has the small intrinsic energy barrier (near 1.5 kcal/mol) characteristic of rapid bimolecular proton transfer between nitrogen and oxygen acids and bases in solution. The observed overall energy barrier (near 10 kcal/mol) for proton transfer in the catalysis indicates the involvement of accompanying energy-requiring processes, such as solvent reorganization or conformational change. Some of this barrier is likely to involve the formation of hydrogen-bonded water in the active-site cavity through the intramolecular proton transfer that occurs.