Metal-directed affinity labelling: Inactivation and inhibition studies of two zinc alcohol dehydrogenases with twelve imidazole derivatives

Abstract

The role of metals in metalloproteins is the object of much research. Metal-directed affinity labels can provide a new way of studying such metals and their near surround~gs, as well as the specific inactivation of met~oenzymes. The general claims for such a reagent are the ability to ligand a metal atom, and the capacity to modify an amino acid residue. A selective modification of amino acid residues in the metalbinding region of the protein may thus be attained. Each subunit of alcohol dehydrogenase from liver contains one structural and one catalytic zinc atom [ 11. The former has four protein ligands while the latter has three to the protein and one free. For the yeast enzyme, ihe exact number of zinc atoms is the subject of discussion [2,3]. The monodentate chelate-binding molecule, imidazole, binds as a free ligand to zinc in the active site of the liver enzyme, and can promote activation or inhibition of the enzyme [4,5]. Imidazole inhibits the yeast enzyme competitively with ethanol (unpublished results). It has been shown that (&Q-2bromo-3(5-imidazolyl) propionic acid (BIP), prior to selective and irreversible alkylation of cysteine-46, binds reversibly at the active site zinc atom of liver alcohol dehydrogenase through the imidazole ring [6,71. The aim of this work has been to survey how a series of metal-d~ected affmity labels react with two metalloenzymes. Twelve imidazole derivatives are surveyed for inhibition and inactivation of two zinc