Affinity Labelling of Yeast and Liver Alcohol Dehydrogenases with the NAD Analogue 4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine

Abstract

The NAD analog 4-(3-bromoacetylpyridinio)butyldiphosphoadenosine inactivates alcohol dehydrogenases [EC 1.1.1.1] from horse liver and yeast by modification of amino acid side chains at the active sites of the proteins. In the presence of excess inactivator the reaction is pseudo 1st order. The stoichiometry is 1 mol inactivator incorporated/mol enzyme subunit. The liver enzyme is inactivated by ketoalkylation of the essential cysteine residue at position 46. No intermediate reactions of other residues are detected, and added cysteine does not influence the modification. In contrast, the labeling results with the yeast enzyme depend on cysteine treatment. The only radioactive peptide isolated is labeled on the essential cysteine residue 43.