Participation of buffer in the catalytic mechanism of carbonic anhydrase

is extremely rapid. Thus, with HCO; as substrate the turnover number of human carbonic anhydrase C is 2.5 X 10’ s-l at pH 7.4 and 2.5’C [l]. This implies that H’ must be transported to the active site at a sufficient rate to react with this frequency. Hence, the direct combination of H& with the active site at pH 7.4 would require a rate constant of at least 6 X 1012 M-’ s-l. This value exceeds that of a diffusioncontrolled reaction by almost 3 orders of magnitude and, therefore, H’ must be delivered to the active site in some other form. The most obvious alternative proton donor is the acidic form of the buffer which is generally present in kinetic turnover experiments. Khalifah [2], Lindskog and Coleman [3], and Prince and Woolley [4] have shown that such a buffermediated proton transfer is theoretically feasible. In this paper we present an experimental test of this hypothesis. We find that carbonic anhydrase C is, indeed, activated by buffers. The effect appears general and has been observed with all the investigated buffer