The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C.

The kinetics of catalysis of COZ hydration by human carbonic anhydrases B and C (EC 4.2.1.1) has been reinvestigated with use of an improved pH indicator stop-flow approach that was checked by studying the uncatalyzed rate of hydration. The Michaelis-Menten parameters were determined between pH 5.8 and 8.8 in noninhibitory buffers. For both isoenzymes K, was independent of pH, whereas V max increased with increasing pH. These results are similar to the findings of others on bovine carbonic anhydrase, but differ from the earlier report of Gibbons and Edsall (J. Biol. Chem., 239, 2539 (1964)) in the finding that K, is pH independent. Despite the differences between the low and high activity forms of carbonic anhydrase, there are close kinetic similarities between them that indicate underlying similarities in active site structure. Imidazole inhibits the B enzyme, apparently competitively, but has no effect on the C enzyme. N-Methyl substitution of imidazole abolishes its inhibitory effect. Nitrous oxide does not inhibit catalysis of COz hydration by either of the two human isoenzymes, or by bovine carbonic anhydrase. The infrared absorption studies of Biepe and Wang (J. Biol. Chem., 243, 2779 (1968)) had indicated that N20 and COz bind competitively to a site identified as the substrate-binding site. The present kinetic results are interpreted as representing a great specificity of carbonic anhydrase for the binding of its substrate CO*. It is proposed that the enzyme-catalyzed hydration of COz requires, not only water activation by a basic group, but also charge neutralization in the transition state by an electron acceptor function. The zinc-bound water in carbonic anhydrase could be involved in both donor and acceptor roles.