Comparison of 18O exchange catalyzed by isoenzymes of carbonic anhydrase.

From the Department ofPharmacology and Therapeutics, University ofFlorida, College ofMedicine, Gainesville, Florida 32610 We compare the effect of buffers on the catalysis by bovine carbonic anhydrase, human carbonic anhydrase C (HCA 0, and human carbonic anhydrase B (HCA B) of two types of ‘“0 exchange. Type I, resulting from the hydration- dehydration reaction, is the exchange of IsO between CO, and water. Type II is the exchange of IsO between ‘*C- containing and ‘“C-containing species of COZ. Imidazole, 2,4-lutidine, and N-methylmorpholine are used as buffers near neutral pH. The buffer dependence of type I exchange in the presence of HCA B is analogous to that observed for HCA C and bovine carbonic anhydrase (Silverman, D. N., and Tu, C. K. (1975) J. Am. Chem. Sot. 97,2263-2269) and is consistent with the hypothesis that buffer-facilitated proton transfer enhances the catalysis in these three forms of car- bonic anhydrase. Type II exchange when catalyzed by HCA C and bovine carbonic anhydrase decreases in rate as buffer concentration increases up to about 5 mM buffer. In con- trast, type II exchange when catalyzed by HCA B does not change appreciably as buffer concentration increases up to 50 mM buffer. These data indicate a significant difference in the possible forms of each enzyme which are involved in proton transfer. Whereas buffer-facilitated proton transfer involving free, unbound bovine carbonic anhydrase is com- patible with the observed data, such a buffer-facilitated proton transfer pathway alone cannot account for type II exchange as catalyzed by HCA B. As one possible hypothesis which is consistent with the data, we suggest that for HCA B proton transfer between the active site and buffer occurs at a rate comparable to or greater than the rate of dissociation of CO, from the active site. Human