Carboxymethylated liver alcohol dehydrogenase. Transient kinetic studies and effect of substrate structure on alcohol oxidation.

1. The rate constants for NADH binding and dissociation for carboxymethylated alcohol dehydrogenase have been determined and compared to those for the native enzyme. 2. Steady-state and transient kinetic experiments have shown that the hydrogen transfer step is rate-determining for oxidation of ethanol by carboxymethylated alcohol dehydrogenase. The rate constant of 0.19 s-1 is considerably slower than that for the native enzyme. 3. The steady-state parameter, V/[E], was obtained for each of a series of alcohols and correlated with the Taft sigma parameter. The linear relationship obtained indicates that the same step, hydrogen transfer, is rate-determining for all the alcohols. The sigma value obtained is the same as for the native enzyme; the implications of this for the mechanism of hydrogen transfer are discussed.     

The major piscine liver alcohol dehydrogenase has class-mixed properties in relation to mammalian alcohol dehydrogenases of classes I and III.

The major alcohol dehydrogenase of cod liver has been purified, enzymatically characterized, and structurally analyzed in order to establish original functions and relationships among the deviating classes of the enzyme in mammalian tissues. Interestingly, the cod enzyme exhibits mixed properties--many positional identities with a class III protein, but functionally a class I enzyme--blurring the distinction among the classes of alcohol dehydrogenase. The two domain interfaces, affected by movements upon coenzyme binding, both exhibit substitutions in a manner thus far unique to the cod enzyme. In contrast, coenzyme-binding residues are highly conserved. At the active site, inner and outer parts of the substrate pocket show different extents of amino acid replacement. In total, no less than 7-10 residues of 11 in the substrate binding pocket differ from those of all the mammalian classes, explaining the substrate specificities. However, the inner part of the substrate pocket is very similar to that of the class I enzymes, which is compatible with the observed characteristics of the cod enzyme: ethanol is an excellent substrate (Km = 1.2 mM) and 4-methylpyrazole is a strong inhibitor (Ki = 0.1 microM). These values are about as low as those typical for the ethanol-active class I mammalian enzyme and do not at all resemble those for class III, for which ethanol is hardly a substrate and pyrazole is hardly an inhibitor. Further out in the substrate pocket, several residues differ from the mammalian classes, affecting large substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Esterase activity of liver alcohol dehydrogenase.

Liver alcohol dehydrogenase is found to possess, in addition to its dehydrogenase and dismutase activities, the ability to hydrolyze octanoate esters at a rate approximately $\text{1}\text{500}\text{–}\text{1}\text{1000}$ of that of the dehydrogenase reaction. The esterase and dehydrogenase activities exhibit an identical isozyme pattern indicating that the same protein catalyzes both reactions. Inhibition studies suggest that the esterase activity presumably shares the catalytic domain with the dehydrogenase activity.     

The physiological role of liver alcohol dehydrogenase

1. Yeast alcohol dehydrogenase was used to determine ethanol in the portal and hepatic veins and in the contents of the alimentary canal of rats given a diet free from ethanol. Measurable amounts of a substance behaving like ethanol were found. Its rate of interaction with yeast alcohol dehydrogenase and its volatility indicate that the substance measured was in fact ethanol. 2. The mean alcohol concentration in the portal blood of normal rats was 0.045mm. In the hepatic vein, inferior vena cava and aorta it was about 15 times lower. 3. The contents of all sections of the alimentary canal contained measurable amounts of ethanol. The highest values (average 3.7mm) were found in the stomach. 4. Infusion of pyrazole (an inhibitor of alcohol dehydrogenase) raised the alcohol concentration in the portal vein 10-fold and almost removed the difference between portal and hepatic venous blood. 5. Addition of antibiotics to the food diminished the ethanol concentration of the portal blood to less than one-quarter and that of the stomach contents to less than one-fortieth. 6. The concentration of alcohol in the alimentary canal and in the portal blood of germ-free rats was much decreased, to less than one-tenth in the alimentary canal and to one-third in the portal blood, but detectable quantities remained. These are likely to arise from acetaldehyde formed by the normal pathways of degradation of threonine, deoxyribose phosphate and beta-alanine. 7. The results indicate that significant amounts of alcohol are normally formed in the gastro-intestinal tract. The alcohol is absorbed into the circulation and almost quantitatively removed by the liver. Thus the function, or a major function, of liver alcohol dehydrogenase is the detoxication of ethanol normally present. 8. The alcohol concentration in the stomach of alloxan-diabetic rats was increased about 8-fold. 9. The activity of liver alcohol dehydrogenase is generally lower in carnivores than in herbivores and omnivores, but there is no strict parallelism between the capacity of liver alcohol dehydrogenase and dietary habit. 10. The activity of alcohol dehydrogenase of gastric mucosa was much decreased in two out of the three germ-free rats tested. This is taken to indicate that the enzyme, like gastric urease, may be of microbial origin. 11. When the body was flooded with ethanol by the addition of 10% ethanol to the drinking water the alcohol concentration in the portal vein rose to 15mm and only a few percent of the incoming ethanol was cleared by the liver.     

Avian alcohol dehydrogenase. Characterization of the quail enzyme, functional interpretations, and relationships to the different classes of mammalian alcohol dehydrogenase

The primary structure of the major quail liver alcohol dehydrogenase was determined. It is a long-chain, zinc-containing alcohol dehydrogenase of the type occurring also in mammals and hence allows judgement of the gene duplications giving rise to the classes of the human alcohol dehydrogenase system. The avian form is most closely related to the class I mammalian enzyme (72-75% residue identity), least related to class II (60% identity), and intermediately related to class III (64-65% identity). This pattern distinguishes the mammalian enzyme classes and separates classes I and II in particular. In addition to the generally larger similarities with class I, the avian enzyme exhibits certain residue patterns otherwise typical of the other classes, including an extra Trp residue, present in both class II and III but not in class I, with a corresponding increase in the UV absorbance. The avian enzyme further shows that a Gly residue at position 260 previously considered strictly conserved in alcohol dehydrogenases can be exchanged with Lys. However, zinc-binding residues, coenzyme-binding residues, and to a large extent substrate-binding residues are unchanged in the avian enzyme, suggesting its functional properties to be related to those of the class I mammalian alcohol dehydrogenases. In contrast, the areas of subunit interactions in the dimers differ substantially. These results show that (a) the vertebrate enzyme classes are of distant origin, (b) the submammalian enzyme exhibits partly mixed properties in relation to the classes, and (c) the three mammalian enzyme classes are not as equidistantly related as initially apparent but suggest origins from two sublevels.     

Cobalt exchange in horse liver alcohol dehydrogenase.

The preparation of metal hybrid species of horse liver alcohol dehydrogenase is made possible by the development of carefully delineated systems of metal in equilibrium metal exchange employing equilibrium dialysis. The conditions which are optimal for the site-specific replacement of the catalytic and/or noncatalytic zinc atoms of the native enzyme by cobalt are not identical with those which are utilized for substitution with 65Zn. Thus, while certain 65Zn hybrids can be prepared by exploiting the differential effects of buffer anions, the cobalt hybrids are generated by critical adjustments in the pH of the dialysate. Factors which may determine the mechanism of metal replacement reactions include acid-assisted, ligand-assisted, and metal-assisted dechelation, steric restriction, and ligand denticity as well as physicochemical properties of the enzyme itself. The spectral characteristics of the catalytic and noncatalytic cobalt atoms reflect both the geometry of the coordination complexes and the nature of the ligands and serve as sensitive probes of these loci in the enzyme.     

Multifunctionality of liver alcohol dehydrogenase. Studies of aldehyde dehydrogenase activity

Kinetic studies of the liver alcohol dehydrogenase catalyzed dehydrogenation of aldehydes were carried out over a wide range of octanal concentrations. The effect of specific inhibitors of liver alcohol dehydrogenase on aldehyde dehydrogenase activity was examined. The results were consistent with a steady-state random mechanism with the formation of the ternary E · NADH octanal complex at low temperatures. This ternary complex becomes inconspicuous at high temperatures. The aldehyde dehydrogenase activity was found to associate with all ethanol-active isozymes. The dual dehydrogenase reactions are catalyzed by the same molecule, presumably in the region of the same domain. However, the two activities respond differently to structural changes.     

Self-association of lyophilized horse liver alcohol dehydrogenase.

The molecular weights of lyophilized and non-lyophilized horse liver alcohol dehydrogenase have been compared by quasi-elastic light scattering, and ultracentrifugation. Whereas the non-lyophilized enzyme has the expected molecular weight of 78 000, the lyophilized enz)me has an initial molecular weight of about 10(6) which increases with time by an endothermic process. This result shows that any physical measurement using lyophilized liver alcohol dehydrogenase to investigate the enzyme mechanism, which relies upon the molecular size, will be invalid.