Horse Liver Alcohol Dehydrogenase

Tryptic digests of the [ 14 C]carboxymethylated derivatives of the three isoenzymes EE, ES and SS of horse liver alcohol dehydrogenase have been compared in “fingerprint” experiments. Eight peptide spots present in the digest of the carboxymethylated EE enzyme were not detected in the digest of the carboxymethylated SS enzyme; and seven spots found in the latter were not discovered in the former. No other differences were noticed. The ES derivative yielded both types of spots but in reduced amounts. It is concluded that the E‐ and S‐types of subunits are very similar and that the ES isoenzyme is a hybrid molecule. From the carboxymethylated SS and ES isoenzymes the S‐chain peptides that differ from their counterparts in the E‐chain were prepared and their structures analysed. They were compared to the known structures [1–3] of the corresponding E‐chain peptides. It is concluded that all the differences between the two sets of peptides are accounted for by amino acid changes at only six positions along the protein chains of the E‐ and S‐types of subunits, and an ancestral geneduplication is suggested. The differences at five positions (17, 94, 101, 110 and 366) are amino acid exchanges compatible with one‐base mutations, while the nature of the sixth difference (position 115) is not fully established. The six differences make the S‐chain more hydrophobic and three units of charge more positive than the E‐chain. These properties fit the solubilities and electrophoretic mobilities of the three isoenzymes. The difference in substrate specificity between the E‐ and S‐chains might be explained by a direct participation in the substrate binding site of some of the residues exchanged, but other explanations cannot be excluded.