IDENTIFICATION OF THE AMINO-ACID RESIDUE MODIFIED IN BACILLUS-STEAROTHERMOPHILUS ALCOHOL-DEHYDROGENASE BY THE NAD+ ANALOG 4-(3-BROMOACETYLPYRIDINIO)BUTYLDIPHOSPHOADENOSINE

4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl‐14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme‐coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C‐labelled coenzyme analogue and subsequently carboxymethylated with unlabelled 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 analogue. This residue is homologous to Cys‐43 in yeast alcohol dehydrogenase and Cys‐46 in the horse liver enzyme but, unlike the latter two, Cys‐38 is not reactive towards iodoacetate in the native bacterial enzyme. Copyright © 1979, Wiley Blackwell. All rights reserved