Basic features of class-I alcohol dehydrogenase: variable and constant segments coordinated by inter-class and intra-class variability. Conclusions from characterization of the alligator enzyme.
The enzymatic and structural properties of alligator liver alcohol dehydrogenase have been determined. Aliphatic and alicyclic alcohols serve as substrates for this first reptilian form of the enzyme characterized, with Km values decreasing rapidly from methanol to hexanol, as for the human class I enzymes, and a Km of 1.2 mM for ethanol at pH 9.9. The N-terminus of the 374-residue protein chain is acetyl-blocked. The enzyme is related in descending order to class I > III > V > II of the structurally characterized mammalian alcohol dehydrogenases. This observation is compatible with the presence of a I/III ancestral line. Differences of the enzyme classes exceed those of the species, suggesting an early origin of the classes. Within its enzyme class, the reptilian protein is most closely related to the avian form (82% residue identities), and is closer to the human than to the amphibian form (76%, versus 69%, respectively). This establishes class I alcohol dehydrogenase as an enzyme having fairly constant rate of change during much of vertebrate evolution, approximately 10% residue differences/100 million years of separation between pairs compared. Residues interacting with the substrate and coenzyme are largely conserved. In the alligator enzyme, there are only four replacements in the substrate pocket compared with the human class I gamma subunit, and those are not known to have functional roles. These properties account for the kinetic parameters, and suggest distinct metabolic functions for the class I enzyme in vertebrates. Comparisons of the enzymes of the different vertebrate lines reveal that segment patterns are characteristic features of the class I enzymes. Three segments are 'variable', while two are 'constant', and both these types of segment are identical with those of the classes. There is extensive variability in close proximity to the active site of the enzyme and this appears to constitute a fundamental property of class I liver alcohol dehydrogenases in general.