Identification of the Amino Acid Residue Modified in Bacillus stearothermophilus Alcohol Dehydrogenase by the NAD+ Analogue 4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine

Abstract

4-(3-Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl-14C]acetyl label. The reactive coenzyme analog inactivates alcohol dehydrogenase from B. stearothermophilus by forming a covalent enzyme-coenzyme compound. The inactivation kinetics and spectral properties of the modified enzyme after treatment with sodium hyposulfite suggest that the analog is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C-labeled coenzyme analog and subsequently carboxymethylated with unlabeled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys-38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analog. This residue is homologous to Cys-43 in yeast alcohol dehydrogense and Cys-46 in the horse liver enzyme but, unlike the latter 2, Cys-38 is not reactive towards iodoacetate in the native bacterial enzyme.