Human Alcohol Dehydrogenase: Structural Differences between the β and γ Subunits Suggest Parallel Duplications in Isoenzyme Evolution and Predominant Expression of Separate Gene Descendants in Livers of Different Mammals

Human alcohol dehydrogenase (ADH; alcohol:NAD+oxidoreductase, EC 1.1.1.1) occurs in multiple forms, which exhibit distinct electrophoretic mobilities and enzymatic properties. The homogeneous isoenzymes β1β1and γ1γ1were isolated from livers of Caucasians with ``typical'' ADH phenotype by double ternary complex affinity chromatography and ion exchange chromatography. The differences between the β1and γ1subunits were determined by structural analysis of all tryptic peptides from the carboxymethylated proteins. The human β1and γ1chains differ at 21 of the 373 positions (5.6%). Ten tryptic peptides account for the differences. All residue substitutions are compatible with one-base mutations and result in largely unaltered properties, but five lead to charge differences. Sixteen substitutions are at positions corresponding to the catalytic domain of the well-known horse enzyme; five correspond to the coenzyme-binding domain. Substitutions adjacent to important regions may correlate with differences in coenzyme binding, substrate specificities, and active-site relationships. The residue replacements between the β1and γ1subunits of human ADH are not identical to the known substitutions between ethanol-active (E) and steroid-active (S) subunits of horse ADH. Thus, the duplication leading to human β1and γ1subunits is separate and different from that leading to equine E and S subunits. Both duplications are likely to have occurred after the ancestral separation of human and equine ADH. Of the 21 residues that are different between β1/γ1, 13 in γ1but only 6 in β1are identical to those of the horse E chain. This suggests a closer relationship between γ1and E, although β1in man and E in the horse are the subunits recovered in highest yield from liver ADH preparations. Consequently, in these two mammalian species, relative activities of genes for an isoenzyme family appear to be different.