Some Characteristics of Human, Bovine, and Horse Carbonic Anhydrases As Revealed by Inactivation Studies

Abstract

Two representatives of the low (human Enzyme B and horse Enzyme B) and two representatives of the high (bovine Enzyme B and human Enzyme C) catalytic activity forms of erythrocyte carbonic anhydrase were reacted with bromoacetate and bromoacetazolamide with the aim of elucidating structural differences at their active sites. The dissociation constants of the reversible inhibition of bromoacetate and bromoacetazolamide have been determined. It was found that the constants for bromoacetate showed 25- to 30-fold differences between the low and high activity forms of the enzyme, but only 1.1- to 6-fold differences within one activity type. The dissociation constants of the enzyme-bromoacetazolamide complex were almost the same for the two groups. Several lines of evidence suggest that bromoacetazolamide inactivates both types of enzymes with simultaneous alkylation of a histidine (or histidines) at or near the active site at the 3-nitrogen position. The rate of alkylation is, however, markedly different for the two types. Stoichiometric amounts of this inhibitor partially alkylate a histidine of the high catalytic activity forms, but do not react with the low catalytic activity forms. At higher inhibitor concentrations both types react, but the high activity forms react significantly faster. By contrast, bromoacetate at low concentrations inactivates exclusively the low catalytic activity forms with the carboxymethylation of a histidine at the 3-nitrogen position. Evidence is presented that this reaction for the horse enzyme occurs at or near the active site. At higher inhibitor concentrations the high catalytic activity forms also became inactivated, but this reaction is nonspecific. The half-times of the inactivation by bromoacetate were also determined. These values for the high catalytic activity enzymes were found to be 36 times greater than could be explained on a basis of enzyme-inhibitor complex concentration. It is suggested that a difference in the conformation of the active site, or a steric hindrance brought about by an amino acid side chain sufficiently close to the reactive histidine, would account for the observed differences in the rates of alkylation and inactivation.