Modification of Alcohol Dehydrogenases with a Reactive Coenzyme Analogue

Nicotinamide–5-bromoacetyl-4-methyl-imidazole dinucleotide was synthesized with and without a 32P or 14C label. This NAD analogue acts as hydrogen acceptor during enzymatic oxidation of ethanol by alcohol dehydrogenases. Due to the reactive bromoacetyl group the analogue also inactivates the enzymes by covalent modification of the proteins. Stoichimetry of binding, spectral properties of binary complexes and enzymatic parameters suggest that the analogue is bound at the coenzyme binding sites of the enzymes, where adjacent residues are alkylated. The residues modified in horse liver and yeast alcohol dehydrogenases were identified after coupling with the 32P-labelled analogue, or with the non-radioactive analogue and subsequent reduction with 3H-labelled sodium borohydride. The labelled proteins were digested with chymotrypsin and the radioactive peptides analyzed. One cysteine residue was specifically modified in each of the two proteins. In the yeast enzyme this was Cys-43 in the tentative sequence, while in the horse protein residues (Cys-46 and Cys-174 in the numbering system of the horse protein) are present in both enzymes, at corresponding but numerically slightly different positions. The reagent thus alkylates alternative residues in these related proteins. The results suggest that Cys-46 and Cys-174 are spatially close together at the active site region of horse liver alcohol dehydrogenase, and that the same applies to corresponding residues in the yeast enzyme. This is in excellent agreement with other data from chemical modifications and X-ray crystallographic analyses.

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