Ligand exchange at the active site of the carbonic anhydrases: a "ping-ping-pong" view.

We emphasize that the interconversion of CO2 and HCO-3 catalyzed by carbonic anhydrase must be regarded as two-substrate, two-product reactions, involving CO2, H2O and HCO-3, H+. Questions then arise regarding the interactions of substrates and products with enzyme during catalysis. Hydration of CO2 has been described by others as a pair of half reactions in which HCO-3 product is released from enzyme, followed by (usually buffer-catalyzed) release of H+. This "ping-pong" view is extended here to a "ping-ping-pong" sequence; based on analyses of specific models for the several intermediate complexes, we conclude that the second "ping" relates to the rate of ligand exchange of H2O from a pentacoordinate enzyme-H2O-HCO-3 complex, about which little is known at present and that is difficult to investigate. We discuss the types of experiments used to measure enzymatic activity and note that (1) they generally measure the rate of the combined "ping-ping" sequence rather than either independently, and (2) the pathway for exchange of substrate H2O is unrelated to the exchange of H2O measured by proton magnetic relaxation and by loss of isotopically labeled oxygen from the CO2-HCO-3 system. The "ping-ping-pong" view, while giving new insights and raising new questions, is consistent with OH- as the ligand of the high-pH form of isozymes I and II, as well as with the results of magnetic relaxation, isotope mixing and loss, 13C NMR linewidths of CO2 and HCO-3 at equilibrium, and stopped-flow kinetic measurements.