Modification of Alcohol Dehydrogenases with a Reactive Coenzyme Analogue

摘要: 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 also inactivates enzymes covalent modification proteins. Stoichimetry binding, spectral properties binary complexes parameters suggest that is bound at coenzyme binding sites enzymes, where adjacent residues are alkylated. The modified in horse liver yeast dehydrogenases were identified after coupling 32P-labelled analogue, non-radioactive subsequent reduction 3H-labelled sodium borohydride. The labelled proteins digested chymotrypsin radioactive peptides analyzed. One cysteine residue specifically each two In enzyme this Cys-43 tentative sequence, while protein (Cys-46 Cys-174 numbering system protein) present both corresponding but numerically slightly different positions. reagent thus alkylates alternative these related proteins. The results Cys-46 spatially close together active site region dehydrogenase, same applies enzyme. excellent agreement other data from chemical modifications X-ray crystallographic analyses.