Irreversible inactivation of bovine carbonic anhydrase B by bromoacetazolamide.

Bromoacetazolamide effects rather quickly a partial, and more slowly an almost complete, irreversible inactivation of bovine carbonic anhydrase B (EC 4.2.1.1). Under identical conditions, the enzyme is not inactivated by bromoacetic acid or iodoacetamide, nor does it react significantly with these compounds. The zinc-free enzyme does not undergo irreversible binding with W-bromoacetazolamide and can be fully reactivated by zinc after exposure to bromoacetazolamide. At low inhibitor concentrations 1 eq of W-bromoacetazolamide reacts with the native enzyme. It is postulated that the enzyme at first forms a complex with the inhibitor. This rapid step is followed by a slower formation of a covalent bond between bromoacetazolamide and an amino acid at or close to the active site of the enzyme resulting in irreversible inactivation. Amino acid analysis of the native and inhibited enzymes revealed that, as a consequence of irreversible inactivation, the 3 nitrogen of 1 eq of histidine had reacted with bromoacetazolamide. At higher inhibitor concentrations, 2 eq of 14C-bromoacetazolamide combined with the enzyme. It is suggested that the initial formation of a complex or covalent bond by 1 molecule of bromoacetazolamide alters the three-dimensional structure of the enzyme so as to make a second combining site available.