Alcohol Dehydrogenase Class III Contrasted to Class I

Abstract

Alcohol dehydrogenases of classes I (the classical liver enzyme) and III (formaldehyde dehydrogenase) constitute a pair of moderately related enzymes (63% residue identity between the human forms) that differ fundamentally in many respects. To elucidate the nature of the differences, we have characterized alcohol dehydrogenase from the most primitive vertebrate line (a cyclostome, Atlantic Hagfish), related that to the multiplicity of the human enzyme, and submitted the enzymes to in vitro hybridization for evaluation of subunit interactions. Three findings illustrate important principles of the enzyme system. First, the alcohol dehydrogenase purified from cyclostomes is a class-III protein, compatible with the facts that cyclostomes constitute the earliest extant vertebrate line and that class III has a distant pre-vertebrate origin. Second, the hagfish enzyme shows multiplicity, with acidic forms in decreasing yield and with amino acid sequences identical between two major isoforms, both aspects constituting properties similar to those of the corresponding human forms. The chemically different subunits are present as homodimers and heterodimers of unmodified and modified subunits, suggesting that the class-III multiplicity derives from modification of a type common to lines as divergent as mammals and cyclostomes. Third, the human enzyme can form cross-species hybrid dimers in vitro with the cod and hagfish or Drosophila class-III enzymes (positional identity with the human form of 82, 76 and 70%, respectively). Hence, the results provide experimental evidence for little class-III divergence in the segments of subunit interactions. The extent of conservation of residues directly involved in the formation of the subunit interface also reveals a clearly different pattern between classes I and III. This highlights separation of divergent forms in an enzyme system, with the constant form (class III) resembling house-keeping enzymes, and exhibiting a correlation between subunit-interacting and substrate-interacting segments.