The kinetics of catalysis of CO2 hydration by human carbonic anhydrases B and C (EC 220.127.116.11) 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 Km was independent of pH, whereas Vmax 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 Km 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 CO2 hydration by either of the two human isoenzymes, or by bovine carbonic anhydrase. The infrared absorption studies of Riepe and Wang (J. Biol. Chem., 243, 2779 (1968)) had indicated that N2O and CO2 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 CO2.
It is proposed that the enzyme-catalyzed hydration of CO2 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.
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