The amino acid sequence of sheep liver sorbitol dehydrogenase has been fitted to the high-resolution model of the homologous horse liver alcohol dehydrogenase by computer graphics. This has allowed construction of a model of sorbitol dehydrogenase that provides explanations why sorbitol is not a substrate for alcohol dehydrogenase, why ethanol is not a substrate for sorbitol dehydrogenase, and what determines its specificity for polyols. An important feature of the model is that one of the ligands to the active site zinc atom is a glutamic acid residue instead of a cysteine residue, which is the corresponding ligand in the homologous alcohol dehydrogenases. This is one component of the structural change that can be related to the different substrate specificities, showing how altered enzymic activity might be brought about by structural changes of the kind that it is now possible to introduce by site-directed mutagenesis and recombinant DNA techniques.

From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Conformational models of the three characterized classes of mammalian liver alcohol dehydrogenase were constructed using computer graphics based on the known three-dimensional structure of the E subunit of the horse enzyme (class 1) and the primary structures of the three human enzyme classes. This correlates the substratebinding pockets of the class I subunits (a, and y in the human enzyme) with those of the class I1 and I11 subunits (z and x, respectively) for three enzymes that differ in substrate specificity, inhibition pattern and many other properties. The substrate-binding sites exhibit pronounced differences in both shape and properties. Comparing human class I subunits with those of class 11 and I11 subunits there are no less than 8 and 10 replacements, respectively, out of 11 residues in the substrate pocket, while in the human class I isozyme variants, only 1 -3 of these 11 positions differ. A single residue, Va1294, is conserved throughout. The liver alcohol dehydrogenases, with different substrate-specificity pockets, resemble the patterns of other enzyme families such as the pancreatic serine proteases.The inner part of the substrate cleft in the class I1 and I11 enzymes is smaller than in the horse class I enzyme, because both Ser48 and Phe93 are replaced by larger residues, Thr and Tyr, respectively. In class 11, the residues in the substrate pocket are larger in about half of the positions. It is rich in aromatic residues, four Phe and one Tyr, making the substrate site distinctly smaller than in the class I subunits. In class Ill, the inner part of the substrate cleft is narrow but the outer part considerably wider and more polar than in the class I and I1 enzymes. In addition, Ser (or Thr) and Tyr in class I1 and I11 instead of His51 may influence proton abstraction/donation at the active site.Mammalian zinc-containing alcohol dehydrogenases constitute an enzyme family of multiple forms. Subunit types a, ,/I and y [l] in dimeric combinations constitute the isozymes of the human class I enzyme [2] and are all homologous to the E subunit of the horse EE isozyme [3], the only alcohol dehydrogenase crystallographically analyzed [4]. The class I1 and I11 enzymes differ considerably in primary structure [5 -81, constituting essentially separate and distinct enzymes [9] with different evolutionary rates [S].Previous model-building studies have shown large similarities between the isozymes within class I and have explained the consequences of the replacements that occur [lo]. These residue exchanges are few but have effects on substrate [lo] and coenzyme [113 binding. The inter-class differences are large and affect charge, enzyme activity, inhibition pattern, and other properties utilized for detection and purification [2]. Ethanol at 5 mM saturates the traditional class I enzymes, while at fhis concentration class I1 contributes less than 15% to the total ethanol oxidation of the liver [12]. Class I11 of human liver alcohol dehydrogenase, with x subunits, is almost inactive towards ethanol and ev...

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