Substrate specificity of human class I alcohol dehydrogenase homo- and heterodimers containing the beta 2 (Oriental) subunit

In order to gain a better understanding of the metabolism of ethanol in Orientals, the kinetic properties of human alcohol dehydrogenase (ADH) isozymes containing the beta 2 (Oriental) subunit, i.e., alpha beta 2, beta 2 gamma 1, beta 2 beta 2, beta 2 gamma 2, as well as gamma 1 gamma 1, were examined by using primary and secondary alcohol substrates of various chain lengths and compared with those of the corresponding beta 1 (Caucasian) subunit containing isozymes already on record [Wagner, F. W., Burger, A. R., & Vallee, B. L. (1983) Biochemistry 22, 1857-1863]. With primary alcohols, these isozymes follow typical Michaelis-Menten kinetics with a preference for long-chain alcohols, as indicated by Km and kcat/Km values. The kcat values obtained with primary alcohols, except methanol, do not vary greatly, i.e., less than 3-fold, whereas the corresponding Km values span a 3600-fold range, i.e., from 26 microM to 94 mM, indicating that the specificity of these isozymes manifests principally in substrate binding. As a consequence, ethanol--which might be thought to be the principal in vivo substrate for ADH--is oxidized rather poorly, i.e., from 50- to 90-fold less effectively than octanol. Secondary alcohol oxidation by the homodimers beta 2 beta 2 and gamma 1 gamma 1 also follows normal Michaelis-Menten kinetics. Again, values of Km and kcat/Km reveal that both isozymes prefer long carbon chains. For all secondary alcohols studied, the Km and kcat values for beta 2 beta 2 are much higher than those for gamma 1 gamma 1, i.e., 25- to 360-fold and 6- to 16-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of thiodiglycol (2,2'-thiobis-ethanol) by alcohol dehydrogenase: comparison of human isoenzymes

Sulfur mustard is a chemical warfare agent that causes blistering of the skin and damages the eyes and airway after environmental exposure. We have previously reported that thiodiglycol (TDG, 2,2'-bis-thiodiethanol), the hydrolysis product of sulfur mustard, is oxidized by alcohol dehydrogenase (ADH) purified from horse liver or present in mouse liver and human skin cytosol. Humans express four functional classes of ADH composed of several different isozymes, which vary in their tissue distribution, some occurring in skin. To help us evaluate the potential contribution of the various human isozymes toward toxicity in skin and in other tissues, we have compared the catalytic activity of purified human class I alphaalpha-, beta1beta1-, beta2beta2-, and gamma1gamma1-ADH, class II pi-ADH, class III chi-ADH, and class IV sigma-ADH with respect to TDG oxidation and their relative sensitivities to inhibition by pyrazole. Specific activities toward TDG were 123, 79, 347, 647, and 12 nmol/min/mg for the class I alphaalpha-, beta1,beta1-, beta2beta2-, and gamma1gamma1-ADH and class II pi-ADH, respectively. TDG was not a substrate for class III chi-ADH. The specific activity of class IV sigma-ADH was estimated at about 1630 nmol/min/mg. 1 mM pyrazole, a potent inhibitor of class I ADH, inhibited the class I alphaalpha, beta1beta1, beta2beta2, and gamma1gamma1 ADH and class IV sigma-ADH by 83, 100, 56, 90, and 73%, respectively. The class I alphaalpha- and beta1beta1-ADH oxidized TDG with kcat/Km value of 7-8 mM(-1) min(-1), beta2beta2-ADH with a value 19 mM(-1) min(-1) and class I gamma1gamma1-ADH with a value of 176 mM(-1) min(-1). The kcat/Km value for class IV sigma-ADH was estimated at 4 mM(-1) min(-1). The activities of class IV sigma-ADH and class I gamma1gamma1-ADH are of significant interest because of their prevalence in eyes, lungs, stomach, and skin, all target organs of sulfur mustard toxicity.     

3 beta-Hydroxy-5 beta-steroid dehydrogenase activity of human liver alcohol dehydrogenase is specific to gamma-subunits

Human liver alcohol dehydrogenase [alcohol:NAD+ oxidoreductase, EC 1.1.1.1 (ADH)] catalyzes the stereospecific oxidation of different 3 beta-hydroxy-5 beta-steroids with ranges of Km from 46 to 320 microM and values of kcat from 7.0 to 72 min-1, pH 8.5. Only the class I isozymes containing gamma-subunits, gamma 1 gamma 1, alpha gamma 1, beta 1 gamma 1, gamma 2 gamma 2, and beta 1 gamma 2, catalyze oxidation of these steroids with kcat/Km ratios 4-10-fold greater than those for ethanol. In marked contrast, class I alpha alpha, alpha beta 1, and beta 1 beta 1, class II, and class III isozymes do not oxidize 3 beta-hydroxy-5 beta-steroids though they readily oxidize ethanol. 1,10-Phenanthroline and 4-methylpyrazole competitively inhibit both alcohol dehydrogenase catalyzed ethanol and 3 beta-hydroxy-5 beta-steroid oxidation demonstrating that the catalysis of both types of substrates occurs at the same active site. The gamma-subunit-catalyzed oxidation of 3 beta-hydroxy-5 beta-steroids is the most specific catalytic function described thus far for any human liver alcohol dehydrogenase isozyme: there is no other isozyme that catalyzes this reaction. Testosterone, an allosteric inhibitor of ethanol oxidation specific for gamma-subunit-containing human liver ADH isozymes [M?rdh, G., Falchuk, K. H., Auld, D. S., & Vallee, B. L. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2836-2840], also noncompetitively inhibits gamma-subunit-catalyzed sterol oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human class I alcohol dehydrogenases catalyze the interconversion of alcohols and aldehydes in the metabolism of dopamine

The class I human liver alcohol dehydrogenases (ADHs) catalyze the interconversion of the intermediary alcohols and aldehydes of dopamine metabolism in vitro, whereas those of the class II and class III do not. The individual, homogeneous class I isozymes oxidize (3,4-dihydroxyphenyl)ethanol and (4-hydroxy-3-methoxyphenyl)ethanol (HMPE) and ethanol with kcat/Km values in the range from 16 to 240 mM-1 min-1 and from 16 to 66 mM-1 min-1, respectively. They reduce the corresponding dopamine aldehydes (3,4-dihydroxyphenyl)acetaldehyde and (4-hydroxy-3-methoxyphenyl)acetaldehyde (HMPAL) with kcat/Km values varying from 7800 to 190,000 mM-1 min-1, considerably more efficient than the reduction of acetaldehyde with kcat/Km values from 780 to 4900 mM-1 min-1. For beta 1 gamma 2 ADH, ethanol competes with HMPE oxidation with a Ki of 23 microM. In addition, 1,10-phenanthroline inhibits HMPE oxidation and HMPAL reduction with Ki values of 20 microM and 12 microM, respectively, both quite similar to that for ethanol, Ki = 22 microM. Thus, both ethanol/acetaldehyde and the dopamine intermediates compete for the same site of ADH, a basis for the ethanol-induced in vivo alterations of dopamine metabolism.     

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives.     

Catalytic properties of human liver alcohol dehydrogenase isoenzymes

Human liver alcohol dehydrogenase (ADH) exists in multiple molecular forms which arise from the association of eight different types of subunits, alpha, beta 1, beta 2, beta 3, gamma 1, gamma 2, pi, and chi, into active dimeric molecules. A genetic model accounts for this multiplicity as products of five gene loci, ADH1 through ADH5. Polymorphism occurs at two loci, ADH2 and ADH3, which encode the beta and gamma subunits. All of the known homodimeric and heterodimeric isoenzymes have been isolated and purified to homogeneity. Analysis of the steady-state kinetic properties and substrate and inhibitor specificities has shown substantial differences in the catalytic properties of the isoenzymes. For example, the Km values for NAD+ and ethanol vary as much as 1,000-fold among the isoenzymes. Some of the differences in catalytic properties can be related to specific amino acid substitutions in the ADH isoenzymes.     

Human liver alcohol dehydrogenases catalyze the oxidation of the intermediary alcohols of the shunt pathway of mevalonate metabolism

Human liver alcohol dehydrogenase (ADH) catalyzes the oxidation of 3,3-dimethylallyl alcohol, the intermediary alcohol of the shunt pathway of mevalonate metabolism. ADH isozymes differ in their activities toward this alcohol in the order gamma 1 gamma 1 greater than gamma 2 gamma 2 approximately alfa alfa greater pi pi approximately beta 2 beta 2 approximately beta 1 beta 1 much greater than chi chi; kcat/Km values are 1.4 x 10(8), 1.9 x 10(7), 1.4 x 10(7), 5.6 x 10(6), 3.6 x 10(6), 1.6 x 10(6) and 2.5 x 10(3) M-1 min-1, respectively. The intermediary alcohols geraniol and farnesol of the proposed branch pathways of mevalonate metabolism are also oxidized by these isozymes with similar relative efficiencies. The genetic determinants of ADH isozymes may contribute to the observed differences in serum cholesterol levels among and within various populations.     

Oxidation and reduction of 4-hydroxyalkenals catalyzed by isozymes of human alcohol dehydrogenase

4-Hydroxyalkenals, natural cytotoxic products of lipid peroxidation, are substrates for human alcohol dehydrogenases (ADH). Class I and II ADHs reduce aliphatic 4-hydroxyalkenals with chain lengths of from 5 to 15 carbons at pH 7 with kcat and Km values comparable to simple aliphatic aldehydes of the same chain length. Class II is particularly effective in the reduction with kcat values as high as 3300 min-1 for 4-hydroxyundecenal. Class III ADH is essentially inactive toward all of these substrates. The class I and II isozymes also catalyze the oxidation of the 4-hydroxy group at pH 10. However, during the reaction, an NAD(+)-dependent irreversible partial inactivation of the alpha beta 1 isozyme is observed which is attributed, with the aid of computer graphics modeling, to selective modification of the alpha subunit. Both ethanol and 1,10-phenanthroline, known to compete with conventional substrates, instantaneously, reversibly, and competitively inhibit 4-hydroxyalkenal reduction and oxidation, indicating that 4-hydroxyalkenals bind at the same site as do conventional substates. The fact that the class II enzyme pi pi-ADH so far is found only in the liver and that the 4-hydroxyalkenals are the best substrates known for this isozyme suggest that it may play a significant role in cellular defenses in the conversion of the cytotoxic aldehydes to the less reactive alcohols.     

Human alcohol dehydrogenases and serotonin metabolism

Human liver alcohol dehydrogenases (ADH) may participate in serotonin (5-hydroxytryptamine) metabolism. Class I and II isozymes catalyze the oxidation of 5-hydroxytryptophol (5-HTOL) with kcat/Km values ranging from 10 to 100 mM-1 min-1 compared to 4-66 mM-1 min-1 for that of ethanol at pH 7.40, 25 degrees C. The product, 5-hydroxyindoleacetaldehyde, was purified as its semicarbazone and identified by mass spectrometry. Ethanol competitively inhibits 5-HTOL oxidation by beta 1 gamma 2 ADH with a Ki of 440 microM, a value similar to the Km of ethanol, 210 microM. The inhibition constants for 1,10-phenanthroline and 4-methylpyrazole are 20 microM and 80 nM respectively, essentially identical to those obtained with ethanol as substrate, 22 microM and 70 nM, respectively. The competition between ethanol and 5-HTOL for ADH can explain observations of ethanol induced changes in serotonin metabolism in vivo.     

Purification and characterization of a new alcohol dehydrogenase from human stomach

Starch gel electrophoresis of homogenates from human stomach mucosa resolves three alcohol dehydrogenase (ADH) forms: the anodic chi-ADH (class III), the cathodic gamma-ADH (class I), and a new form of slow cathodic mobility that has not been previously characterized. In this work, we describe the purification in three chromatographic steps and the physical and kinetic characterization of this new human alcohol dehydrogenase, which we have named sigma-ADH. The enzyme exhibits the general physicochemical features (Mr, zinc content, subunit Mr, cofactor preference) of all mammalian alcohol dehydrogenases. The kinetic studies show a high Km value (41 mM) and a high kcat value (280 min-1) for ethanol at pH 7.5. The Km decreases as the alcohol increases its chain length. The aldehydes are better substrates than the corresponding alcohols, with m-nitrobenzaldehyde being the best substrate examined. sigma-ADH is strongly inhibited by 4-methylpyrazole, but with a Ki (10 microM) still higher than that for a class I isoenzyme. These properties suggest that sigma-ADH is a class II isoenzyme, different from pi-ADH and similar to that previously described by us in rat stomach. At the high ethanol concentrations in stomach after drinking, sigma-ADH is probably the ADH form with the largest contribution to human gastric ethanol metabolism.     

Characterization of three isoenzymes of rat alcohol dehydrogenase. Tissue distribution and physical and enzymatic properties

Rat tissues contain three different isoenzymes of alcohol dehydrogenase (ADH) that we have named ADH-1, ADH-2 and ADH-3, ADH-1 is an anodic isoenzyme present in high amounts in the ocular tissues, stomach and lung. ADH-2 is also anodic and has been found in all the rat organs examined. ADH-3 is the group of cathodic ADH forms, mainly present in liver, that has been the subject of the majority of the previous studies on rat ADH. The three isoenzymes have been purified to homogeneity and characterized. All of them have similar physical characteristics: Mr 80,000, with two subunits of Mr 40,000; they contain four atoms of Zn per molecule, and prefer NAD+ as cofactor. Isoelectric points are, however, different: 5.1 for ADH-1, 5.95-6.3 for ADH-2 and 8.25-8.4 for ADH-3. ADH-3 exhibits a Km for ethanol of 1.4 mM, a broad substrate specificity and is strongly inhibited by pyrazole (Ki = 0.4 microM). ADH-2 shows substrate specificity toward long-chain alcohols and aldehydes, cannot be saturated by ethanol and is practically insensitive to pyrazole (Ki = 78.4 mM). ADH-1 has intermediate properties, with a Km for ethanol of 340 mM, a broad substrate specificity and Ki for pyrazole of 0.56 mM. Rat ADH-1, ADH-2 and ADH-3 exhibit many analogies with human ADH classes II, III and I respectively. The specific localization and kinetic properties of rat ADH isoenzymes suggest that ADH-1 and ADH-3 may act as metabolic barriers to external alcohols and aldehydes whereas ADH-2 may have a function in the metabolism of the endogenous long-chain alcohols and aldehydes.     

Physical and enzymatic properties of a class III isozyme of human liver alcohol dehydrogenase: chi-ADH

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.     

Characterization of Coturnix quail liver alcohol dehydrogenase enzymes

Livers from male or female Coturnix quail possess up to four electrophoretically distinct bands of alcohol dehydrogenase (ADH) activity. Three pyrazole-sensitive bands of enzymatic activity, designated ADH-1, ADH-2, and ADH-3, are cathodic at pH 8.2, and the fourth, ADH-An, is neutral to slightly anodic and insensitive to pyrazole. ADH-2 and ADH-3, and occasionally ADH-1, are present in livers from immature females. The predominant enzyme in immature male livers is ADH-3. At sexual maturity all three pyrazole-sensitive enzymes are present in livers from male birds, and livers from females possess predominantly ADH-3. ADH-2 and ADH-3, purified from female livers, are dimers of 80,000 daltons possessing 4 mol of Zn2+/mol of native protein. Both ADH-2 and ADH-3 were inhibited by 4-methylpyrazole with KI values of 430 and 335 nM, respectively. These values are similar to those of human class I isoenzymes. Neither enzyme oxidized methanol or ethylene glycol, which distinguished them from mammalian pyrazole-sensitive ADH isoenzymes. Both ADH-2 and ADH-3 showed specificity toward hydrophobic primary alcohols and were most active toward benzyl alcohol and n-octanol.     

A human liver alcohol dehydrogenase enzyme-linked immunosorbent assay method specific for class I, II, and III isozymes

A sensitive and convenient method for the quantitative measurement of human alcohol dehydrogenase (ADH) isozymes based on enzyme-linked immunosorbent assay has been devised. The procedure was optimized with respect to antigen coating density, antiserum dilution, and incubation times with rabbit antisera raised against beta 1 beta 1-ADH to achieve a limit of sensitivity of 1 ng/ml for this isozyme when purified. Using the optimal conditions established, quantitative measurement of alpha beta 1, alpha gamma 1, beta 1 gamma 1, pi, and chi-ADH were obtained with antisera raised in rabbits toward these individual isozymes. The incorporation into the procedure of thimerosal (ethyl(4-mercaptobenzoato-S)mercury) or other sulfhydryl specific reagents improved the soluble phase antiserum avidity for all ADH isozymes, thereby increasing the sensitivity. Thimerosal is an absolute requirement for chi-ADH antigen-antibody binding. The polyclonal rabbit antisera elicited by the individual isozymes of the three classes of ADH exhibit a high degree of isozyme class specificity. Cross-reactivity of the antibodies with the beta 1 beta 1, alpha gamma 1, alpha gamma 2, alpha beta 1, beta 1 gamma 1, beta 1 gamma 2, pi and chi isozymes were evaluated. Antisera against the class I isozymes beta 1 beta 1 and beta 1 gamma 1 cross-react with all class I isozymes and with pi-ADH. Antibodies against pi and chi-ADH are selective and specific only for their respective antigens. Neither one cross-reacts with any class I isozyme. Conformational effects resulting from subunit interactions likely account for differences in cross-immunoreactivity between the closely homologous class I isozymes.     

Kinetic and electrophoretic properties of native and recombined isoenzymes of human liver alcohol dehydrogenase

Ten, electrophoretically distinct, molecular forms of alcohol dehydrogenase have been isolated from a single human liver by affinity and ion-exchange chromatography. The starch gel electrophoresis patterns after the dissociation-recombination of the forms are consistent with the hypothesis that they arise from the random combination of alpha, beta 1, gamma 1, and gamma 2 subunits into six heterodimeric and four homodimeric isoenzymes. Large differences in kinetic properties are observed for the homodimeric isoenzymes, alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. At pH 7.5, the Km value of beta 1 beta 1 for ethanol is 0.049 mM and that of alpha alpha is 4.2 mM. Forms gamma 1 gamma 1 and gamma 2 gamma 2 do not obey Michaelis-Menten kinetics at pH 7.5 but exhibit negative cooperativity with Hill coefficients of 0.54 and 0.55 and [S]0.5 values of 1.0 and 0.63 mM, respectively. However, all isoenzymes display Michaelis-Menten kinetics for ethanol oxidation at pH 10.0 with Km values ranging from 1.5 to 3.2 mM. The maximum specific activity of beta 1 beta 1 is considerably lower than that of the other three homodimers at both pH 7.5 and 10.0. The Km values of the four homodimers for NAD+ at pH 7.5 range from 7.4 to 13 microM and those for NADH, from 6.4 to 33 microM. Ki values for NADH range from 0.19 to 1.6 microM. At pH 7.5, the kinetic properties of alpha alpha and beta 1 beta 1, prepared in vitro from dissociated and recombined alpha beta 1, are similar to those of the native homodimers. The forms gamma 1 gamma 1 and gamma 2 gamma 2, prepared from dissociated and recombined alpha gamma 1 and beta 1 gamma 2, respectively, exhibit negative cooperativity with Hill coefficients that are similar to those seen with the respective native homodimers.     

Effect of selected alcohol dehydrogenase inhibitors on the human heart lactate dehydrogenase activity--an in vitro study

Metabolic acidosis complicates methanol, ethylene glycol and other alcohol intoxications. It is caused firstly by acid metabolites and secondly by the lactate elevation. The aim of the study was to evaluate the effect of alcohol dehydrogenase (ADH; EC 1.1.1.1) inhibitors and substrates: 4-methylpyrazole (4-MP), cimetidine, EDTA, ethanol and methanol on lactate dehydrogenase (LDH; EC 1.1.1.27) activity. The activity of LDH was determined spectrophotometrically in in vitro human heart homogenates with the mentioned compounds at 0.01, 0.1, 1.0 mM concentrations of 4-MP, cimetidine, EDTA, and 12.5, 25.0, 50.0 mM of ethanol and methanol. The LDH activity was significantly inhibited by 0.1 mM (p<0.05) and 1.0 mM (p<0.01) 4-MP and 1.00 mM EDTA (p<0.05). Higher LDH activity vs. control was observed in the samples incubated with all studied ethanol and methanol concentrations but these differences were not statistically significant. Thus, 4-MP was found to be the most effective inhibitor of LDH of all compounds tested. Therefore, such effect of 4-MP seems to be an additional advantage in methanol and ethylene glycol intoxications.     

New human liver alcohol dehydrogenase forms with unique kinetic characteristics

All adult and infant human liver homogenates studied thus far show two previously unreported forms of alcohol dehydrogenase on starch gel electrophoresis. Under the conditions employed, these forms migrate toward the anode and readily stain for pentanol but virtually not for ethanol oxidizing activity. In contrast, all human ADH isoenzymes identified previously are cathodic and react equally well with either substrate. These new ADH forms have been separated from the other known ones by DEAE-cellulose chromatography and are then purified on Agarose-hexane-AMP. Although the physical characteristics of the new anodic ADH forms are similar to those of the known human ADH isoenzymes, the former are not inhibited by 12 mM 4-methyl pyrazole, oxidize ethanol very poorly and appear to prefer longer chain alcohols as substrates.     

Class II alcohol dehydrogenase (ADH2)--adding the structure

Class II alcohol dehydrogenase (ADH2) represents a highly divergent class of alcohol dehydrogenases predominantly found in liver. Several species variants of ADH2 have been described, and the rodent enzymes form a functionally distinct subgroup with interesting catalytic properties. First, as compared with other ADHs, the catalytic efficiency is low for this subgroup. Second, the substrate repertoire is unique, e.g. rodent ADH2s are not saturated with ethanol as substrate, and while omega-hydroxy fatty acids are common substrates for the human ADH1-ADH4 isoenzymes, including ADH2, these compounds function as inhibitors rather than substrates. The recently determined structure of mouse ADH2 reveals a novel substrate-pocket topography that accounts for the observed substrate specificity and may, therefore, be important for the exploration of orphan substrates of ADH2. It is possible to improve the catalytic efficiency of mouse ADH2 by an array of mutations at position 47. Residue Pro47 of the wild type ADH2 enzyme seems to strain the binding of coenzyme, which prevents a close approach between the coenzyme and substrate for efficient hydrogen transfer. Based on crystallographic and mechanistic investigations, the effects of residue replacements at position 47 are multiple, affecting the distance for hydride transfer, the pK(a) of the bound alcohol substrate as well as the affinity for coenzyme.     

Identification of a human stomach alcohol dehydrogenase with distinctive kinetic properties

A new form of alcohol dehydrogenase, designated mu-alcohol dehydrogenase, was identified in surgical human stomach mucosa by isoelectric focusing and kinetic determinations. This enzyme was anodic to class I (alpha, beta, gamma) and class II (pi) alcohol dehydrogenases on agarose isoelectric focusing gels. The partially purified mu-alcohol dehydrogenase, specifically using NAD+ as cofactor, catalyzed the oxidation of aliphatic and aromatic alcohols with long chain alcohols being better substrates, indicating a barrel-shape hydrophobic binding pocket for substrate. mu-Alcohol dehydrogenase stood out in high Km values for both ethanol (18 mM) and NAD+ (340 microM) as well as in high Ki value (320 microM) for 4-methylpyrazole, a competitive inhibitor for ethanol. mu-Alcohol dehydrogenase may account for up to 50% of total stomach alcohol dehydrogenase activity and appeared to play a significant role in first-pass metabolism of ethanol in human.