No abstract

Apoenzyme prepared by removal of the 2 mol of Zn2+/mol from Aeromonas aminopeptidase is inactive. Addition of Zn2+ reactivates it completely, and reconstitution with Co2+, Ni2+, or Cu2+ results in a 5.0-, 9.8-, and 10-fold more active enzyme than native aminopeptidase, respectively. Equilibrium dialysis and spectral titration experiments with Co2+ confirm the stoichiometry of 2 mol of metal/mol. The addition of only 1 mol of metal/mol completely restores activity characteristic of the particular metal. Interaction between the two sites, however, causes hyperactivation; thus, addition of 1 mol of Zn2+/mol subsequent to 1 mol of Co2+, Ni2+, or Cu2+ per mole increases activity 3.2-, 42-, or 59-fold, respectively. The cobalt absorption spectrum has a peak of 527 nm with a molar absorptivity of 53 M-1 cm-1 for 1 mol of cobalt/mol, which increases to 82 M-1 cm-1 for a second cobalt atom and is unchanged by further addition of Co2+. Circular dichroic (CD) and magnetic CD spectra indicate that the first Co2+ binding site is tetrahedral-like and that the second is octahedral-like. Stoichiometric quantities of 1-butylboronic acid, a transition-state analogue inhibitor of the enzyme [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322], profoundly affects absorption, CD, and MCD spectra, but n-valeramide, a substrate analogue inhibitor, has no effect. These findings suggest that the tetrahedral-like site is catalytic and the other octahedral-like site is regulatory or structural.

A series of depsipeptides whose structures complement exactly those of oligopeptide substrates studied previously has been examined to explore the dual specificity of carboxypeptidase A. They have the general form R-(Gly)"-L-OPhe where OPhe is phenyllactate and R is benzoyl, benzyloxycarbonyl, or 5-dimethylaminonaphthalene-

chi-Alcohol dehydrogenase (chi-ADH), a class III isozyme characterized by its anodic electrophoretic mobility and lack of inhibition by 4-methylpyrazole, has been isolated from human liver and purified to homogeneity in a reducing medium. chi-ADH resembles other human liver ADH isozymes of classes I and II with respect to its molecular weight, dimeric structure, stoichiometry of zinc and NADH binding, and pH optima for the oxidation of alcohols. This homodimer exhibits subtle differences in its absorption spectrum and amino acid composition relative to those of other human isozymes but differs markedly from their specificity toward alcohols and aldehydes. chi-ADH oxidizes ethanol very poorly. The reaction is bimolecular, and an apparent Km cannot be discerned up to 2.3 M ethanol. The enzyme is inactive toward methanol, ethylene glycol, digitoxigenin, digoxigenin, and gitoxigenin , but alcohols with carbon chain lengths greater than four are oxidized rapidly with Km values decreasing with increasing carbon chain length. Taken jointly, the composition, structure, and enzymatic properties of the ADH isozymes purified and studied so far strongly imply that their metabolic roles, yet to be discovered, will give a new perspective to ethanol metabolism and pathology.

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...