Horse Liver Alcohol Dehydrogenase

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Abstract
The [14C]carboxymethylated protein of the ethanol‐active isoenzyme of horse liver alcohol dehydrogenase has been treated with trypsin (with and without previous maleylation of the substrate), chymotrypsin, pepsin and cyanogen bromide, respectively. Peptide mixtures obtained have been fractionated and analysed. Data are given for those peptides originating from a C‐terminal region comprising about 60% of the protein chain, and the amino acid sequence of this segment, containing the last 234 residues, is deduced. The N‐terminal part was known previously and the primary structure of the whole protein chain is therefore established. The ethanol‐active isoenzyme is found to be a dimer of two completely identical protein chains, each 374 residues long. The N‐terminal part of the molecule contains the reactive cysteine residue, six out of the seven histidine residues in the whole chain, the majority of the mutations between the isoenzyme chains of different substrate specificities, and a region homologous to another dehydrogenase. Serine residues that reacted during maleylation of the protein were found to undergo O → N maleyl shift when they became N‐terminal residues in peptides. Evidence was obtained suggesting that carboxymethylmethionyl peptide bonds are labile.