Crystallization and preliminary crystallographic studies of Saccharomyces cerevisiae alcohol dehydrogenase I.

The cytoplasmic yeast alcohol dehydrogenase I crystallized at 5 degrees C as hexagonal plates or short columns in the presence of NAD+ and 2,2,2-trifluoroethanol, in sodium N-tris(hydroxymethyl)methyl-3-aminopropanesulfonate buffer at pH 8.2 to 8.6, using polyethylene glycol 4000 as precipitant. X-ray diffraction data to 3.2 A resolution show that the crystals are hexagonal in space group P622 with unit cell dimensions a = b = 147.9 A, c = 69.1 A. There is one subunit of the tetrameric enzyme per asymmetric unit, giving a packing density of 2.9 A3/Da.

Inhibition by carboxamides and sulfoxides of liver alcohol dehydrogenase and ethanol metabolism.

Sulfoxides and amides were tested as inhibitors of liver alcohol dehydrogenase and ethanol metabolism in rats. With both series of compounds, increasing the hydrophobicity resulted in better inhibition, and introduction of polar groups reduced inhibition. Of the cyclic sulfoxides, tetramethylene sulfoxide was the best inhibitor as compared to the tri- and pentamethylene analogue and other compounds, and it may be a transition-state analogue. The most promising compounds, tetramethylene sulfoxide and isovaleramide, were essentially uncompetitive inhibitors of purified horse and rat liver alcohol dehydrogenases with respect to ethanol as substrate. These compounds also were uncompetitive inhibitors in vivo, which is advantageous since the inhibition is not overcome at higher concentrations of ethanol, as it is with competitive inhibitors, such as pyrazole. The uncompetitive inhibition constants for tetramethylene sulfoxide and isovaleramide for rat liver alcohol dehydrogenase were 200 and 20 microM, respectively, in vitro, whereas in vivo the values were 340 and 180 mumol/kg. The differences in the values may be due to metabolism or distribution of the compounds. Further studies will be required to determine if isovaleramide or tetramethylene sulfoxide is suitable for therapeutic purposes.

Inactivation of liver alcohol dehydrogenases and inhibition of ethanol metabolism by ambivalent active-site-directed reagents.

Active-site-directed reagents, of the general structure omega-(BrCH2CONH)RCOY, where R = alkyl, aryl, or aralkyl, and Y = OH or NH2, inactivated horse, mouse, rat, and human liver alcohol dehydrogenases at widely different rates, reflecting differences in reagent specificity and in the structures of the enzymes. Treatment of mice and rats with either of two optimally specific reagents, p-(XCH2CONH)C6H4(CH2)3CONH2, where X = Br (7) or CH3SO3 (10), partially (20 to 40%) inactivated alcohol dehydrogenase in liver, inhibited ethanol metabolism, and prolonged the impairment of coordination produced by ethanol in these animals. Although the dose of 7 used (0.13 mmol/kg) approximated the LD50, 10 was effective at a dose of 0.48 mmol/kg that was not acutely toxic.

3-Substituted pyrazole derivatives as inhibitors and inactivators of liver alcohol dehydrogenase.

3-Substituted pyrazoles, HOCH2 (1), HOCH2CH2 (2), HOCH2CH2CH2 (3), ClCH2 (4), ClCH2CH2 (5), ClCH2CH2CH2 (6), and CH3CO (7), were synthesized and evaluated in vitro on horse liver alcohol dehydrogenase for their potential as inhibitors of ethanol metabolism. 1 to 6 bound to the enzyme-NAD+ complex with dissociation constants of 40 to 200 microM, much higher than the constants for the corresponding 4-substituted pyrazoles, but with the same absorption maximum at 295 nm. 4 inactivated the enzyme within a few minutes, but NAD+ protected against reaction, and 4 nonspecifically alkylated many sulfur atoms in the protein. The isomer, 4-(chloromethyl)pyrazole, behaved similarly, 5 and 6 strongly inhibited the enzyme in the presence of NAD+, due to formation of the slowly dissociable (10(-3)s-1) enzyme-NAD+-pyrazole complex, but did not irreversibly inactivate the enzyme. 7 inhibits the enzyme weakly (Kp = 5 mM). It appears that the 3-substituted pyrazoles bind to the enzyme-NAD+ complex with the reactive functional group improperly positioned for specific irreversible reaction.

Sulfonate analogues of adenosine nucleotides as inhibitors of nucleotide-binding enzymes.

2-(Adenin-9-yl)ethanesulfonic acid (1), 3-(adenin-9-yl)propanesulfonic acid (2), 9-(5-deoxy-beta-D-ribofuranosyl)-adenine-5'-sulfonic acid (3), and 9-(3-deoxy-beta-D-arabinofuranosyl)adenine-3'-sulfonic acid (4) were prepared by reaction of the corresponding chlorides by sodium sulfite (1-3) or by reaction of an epoxide with sodium hydrogen sulfite (4). They inhibited a typical nucleotide-binding enzyme, horse liver alcohol dehydrogenase, with inhibition constants in the range of 0.18-4.9 mM at pH 8, 25 degrees C.

Inhibition of human alcohol dehydrogenases by formamides.

Human alcohol dehydrogenase (HsADH) comprises class I (alpha, beta, and gamma), class II (pi), and class IV (sigma) enzymes. Selective inhibitors of the enzymes could be used to prevent the metabolism of alcohols that form toxic products. Formamides are unreactive analogues of aldehydes and bind to the enzyme-NADH complex [Ramaswamy, S.; Scholze, M.; Plapp, B. V. Biochemistry 1997, 36, 3522-3527]. They are uncompetitive inhibitors against varied concentrations of alcohol, and this makes them effective even with saturating concentrations of alcohols. Molecular modeling led to the design and synthesis of a series of cyclic, linear, and disubstituted formamides. Evaluation of 23 compounds provided structure-function information and selective inhibitors for the enzymes, which have overlapping but differing substrate specificities. Monosubstituted formamides are good inhibitors of class I and II enzymes, and disubstituted formamides are selective for the alpha enzyme. Selective inhibitors, with Ki values at pH 7 and 25 degrees C of 0.33-0.74 microM, include N-cyclopentyl-N-cyclobutylformamide for HsADH alpha, N-benzylformamide for HsADH beta1, N-1-methylheptylformamide for HsADH gamma2, and N-heptylformamide for HsADH sigma and HsADH beta1.

Inhibition of liver alcohol dehydrogenase and ethanol metabolism by 3-substituted thiolane 1-oxides.

3-Substituted thiolane 1-oxides (methyl, n-butyl, n-hexyl, and phenyl) were prepared and tested as inhibitors of horse, monkey, and rat liver alcohol dehydrogenases and of ethanol metabolism in rats. These compounds inhibit alcohol oxidation in an uncompetitive manner with respect to ethanol as a varied substrate. Lengthening the alkyl substituent increased the inhibitory potency because of tighter binding in the hydrophobic substrate binding pocket of the alcohol dehydrogenases. Thus, the 3-hexyl derivative was the most potent inhibitor of the purified rat liver alcohol dehydrogenase, with a Kii value of 0.13 microM. The 3-butyl derivative was the best inhibitor of ethanol metabolism in rats, with a Kii value of 11 mumol/kg. The acute toxicity in mice of the butyl derivative was 1.4 mmol/kg. Since high concentrations of alcohol do not prevent the inhibitory effects of these compounds, they may be particularly useful for preventing poisoning by methanol or ethylene glycol.

Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies.

Control of alcohol metabolism.

The rate of alcohol metabolism is determined by the kinetic characteristics and concentrations of the alcohol and aldehyde dehydrogenases and by the rate of restoration of the redox state of the cell. Several potent competitive and uncompetitive inhibitors of the alcohol dehydrogenases can decrease the rate of alcohol metabolism; they may be useful for preventing the potentially deleterious effects of ethanol metabolism. Alcohol dehydrogenases have very broad specificity and can readily reduce a variety of carbonyl compounds by exchange reactions while ethanol is metabolized. Agents that increase the rate of metabolism need to be developed.

Crystallographic investigations of alcohol dehydrogenases.

The structures of horse liver alcohol dehydrogenase class I in its apoenzyme form and in different ternary complexes have been determined at high resolution. The complex with NAD+ and the substrate analogue pentafluorobenzyl alcohol gives a detailed picture of the interactions in an enzyme-substrate complex. The alcohol is bound to the zinc and positioned so that the hydrogen atom can be directly transferred to the C4 atom of the nicotinamide ring. The structure of cod liver alcohol dehydrogenase with hybrid properties (functionally of class I but structurally overall closer to class III) has been determined by molecular replacement methods to 3 A resolution. Yeast alcohol dehydrogenase has been crystallized, and native data have been collected to 3 A resolution.

Mouse alcohol dehydrogenase 4: kinetic mechanism, substrate specificity and simulation of effects of ethanol on retinoid metabolism.

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37 degrees C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD(+) is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K(m) for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.

Rate-limiting steps in ethanol metabolism and approaches to changing these rates biochemically.

Ethanol is oxidized to acetate primarily by a system involving liver alcohol and aldehyde dehydrogenases coupled with reoxidation of NADH by the mitochondria. All of these steps are at least partially rate-limiting in ethanol metabolism, with alcohol dehydrogenase and oxidative phosphorylation probably slower than the others. More research is required to assess the quantitative roles of various steps. Many agents are ineffective in changing the rate of metabolism of ethanol, but fructose and dinitrophenol may increase the rate by up to 1.5-fold in vivo. The failure of single agents to increase the rate substantially may indicate that when one step is accelerated, another step becomes rate-limited. Therefore, combinations of agents that affect several steps simultaneously may be required for acceleration. Effective experimental methods for inhibiting alcohol dehydrogenase in vivo are available.

Kinetics and control of alcohol oxidation in rats.

The rates of oxidation of ethanol and isopropanol by purified rat liver alcohol dehydrogenase were determined in vitro and compared to the rates of metabolism in vivo in order to estimate the extent to which alcohol dehydrogenase activity limits ethanol metabolism. The metabolism of isopropanol and isopropanol-d7 (CD3CDOHCD3) was examined by measuring blood alcohol and acetone levels at various times and apparently proceeds by an irreversible, enzyme-catalyzed pathway: isopropanol leads to acetone leads to an unidentified metabolite. The kinetic constants for the metabolism were computed from simultaneous fits to the appropriate differential equations using a nonlinear least-squares program. The relative rates of oxidation of the alcohols, ethanol:isopropanol:isopropanol-d7, at 25 mM were 9.6 : 2.3 : 1.0 in vitro and 4.1 : 2.4 : 1.0 in vivo. Since the ratio of rates for isopropanol is about the same in vitro and in vivo, it appears that alcohol dehydrogenase activity is the predominant rate-limiting factor in isopropanol metabolism. The relatively slower rate of ethanol oxidation in vivo as compared to in vitro suggests that liver alcohol dehydrogenase is partially (about 40%) limiting for ethanol metabolism.

In vivo evaluation of ambivalent active-site-directed inactivators of liver alcohol dehydrogenase.

In order to decrease the rate of ethanol metabolism for the treatment of acute and chronic alcoholism it would be useful to inhibit liver alcohol dehydrogenase in vivo. Based on a knowledge of the three-dimensional structure of the horse enzyme, we designed active-site-directed inactivators [p-(XCH2CONH)C6H4(CH2)3COHN2] which bind to the enzyme-NAD or enzyme-NADH complex and alkylate methionine residue 306. In vitro, these reagents inactivated mouse, rat, horse and human liver alcohol dehydrogenases faster in the presence than in the absence of NAD or NADH, but with slightly different specificity. Mice and rats pretreated with the reagents eliminated ethanol in blood more slowly than those not treated, and the specific activity of alcohol dehydrogenase in liver homogenates of treated animals was decreased. It appears that the design of active-site-directed reagents is feasible, but these reagents must be improved so that they are more efficacious in vivo.

Catalysis by yeast alcohol dehydrogenase.

Table 7 presents a brief summary of the effects of various mutations on some of the relevant kinetic constants. The results illustrate several important features of the use of site-directed mutagenesis in exploring structure and function of enzymes. Note that most of the mutations affect a given step or kinetic parameter in the mechanism, such as the binding of NAD+ or the turnover number with ethanol. Furthermore, one mutation can affect many steps in the mechanism. Thus, it is difficult to ascribe a particular role to an amino acid residue. It is also difficult to quantify the function of a residue, since the magnitudes of the effects on kinetic parameters will be modulated by the other amino acid residues that participate in the reaction. Comprehensive and quantitative kinetic studies of many mutant enzymes are required if we are to understand catalysis and specificity. We are reluctant to describe any residue as "essential" for activity since substitution with some amino acid can probably produce an enzyme with some residual activity. (Maybe the Thr48Gly enzyme would be active, as a water molecule could substitute for the hydroxyl of the threonine.) Likewise, when substitution of a residue partially, but not totally, decreases activity, it does not necessarily mean that the residue is "not essential". The change in activity can reflect the contribution of that residue to catalysis. On the other hand, if various substitutions of a residue do not change activity, it would be reasonable to conclude that the residue is not essential (Plapp et al., 1971). Most of the amino acid residues at the active site are involved in the catalytic mechanism, either by contacting the substrates directly or by participating in the chemistry. Some of the residues that are outside of the active site are indirectly involved, by affecting the structure of the protein. Substitution of an important amino acid residue should significantly affect activity, and studies on the kinetics and structure should allow one to distinguish among the various explanations.