Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.1 (alcohol dehydrogenase)
9,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of 13C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. alpha,alpha-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD+ balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in AdhF larvae. In AdhS larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic flux may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.
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PMID:Drosophila alcohol dehydrogenase polymorphism and carbon-13 fluxes: opportunities for epistasis and natural selection. 798 61

We previously reported that the D39N mutant of Drosophila alcohol dehydrogenase (ADH), in which Asp-39 is replaced with asparagine, has a 60-fold increase in affinity for NADP+ and a 1.5-fold increase in kcat compared to wild-type ADH [Chen et al. (1991) Eur. J. Biochem. 202, 263-267] and proposed that this part of ADH is close to the 2'-phosphate on the ribose moiety of NADP+. Here we report the effect of replacing Ala-46 with an argine residue, and A46R mutant, on binding of NADP+ to ADH and its catalytic efficiency with the NADP+ cofactor, and a modeling of the three-dimensional structure of the NAD(+)-binding region of ADH. The A46R mutant has a 2.5-fold lower Km(app)NADP+ and a 3-fold higher kcat with NADP+ compared to wild-type ADH; binding of NAD+ to the mutant was unchanged and kcat with NAD+ was lowered by about 30%. For the A46R mutant, the ratio of kcat/Km of NAD+ to NADP+ is 85, over ten-fold lower than that for wild-type ADH. Our model of the 3D structure of the NAD(+)-binding region of ADH shows that Ala-46 is over 10 A from the ribose moiety of NAD+, which would suggest that there is little interaction between this residue and NAD+ and explain why its mutation to arginine has little effect on NAD+ binding. However, the positive charge at residue 46 can neutralize some of the coulombic repulsion between Asp-39 and the 2'-phosphate on the ribose moiety of NADP+, which would increase its affinity for the A46R mutant. We also constructed a double mutant, D39N/A46R mutant, which we find has a 30-fold lower Km(app)NADP+ and 8-fold higher kcat with NADP+ as a cofactor compared to wild-type ADH; binding of NAD+ to this double mutant was lowered by 5-fold and kcat was increased by 1.5-fold. As a result, kcat/Km for the double mutant was the same for NAD+ and NADP+. The principle effect of the two mutations in ADH is to alter its affinity for the nucleotide cofactor; kcat decreases slightly in A46R with NAD+ and remains unchanged or increases in the other mutants.
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PMID:Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP+. 798 26

A high-resolution crystal structure is not currently available for Drosophila alcohol dehydrogenase. A detailed three-dimensional model for this enzyme, based on the structure of 3 alpha,20 beta-hydroxysteroid dehydrogenase, has been generated by extensive computer modeling studies. Aspects of the model concerned with coenzyme binding have been tested by site-directed mutagenesis of residues Gly-14 to Ala, Gly-19 to Ala, Asp-38 to Ala, and Pro-214 to Ser. All enzymes have been characterized in terms of kinetic constants, relative stabilities to guanidinium chloride, and heat inactivation. The contribution of NAD binding to the stabilization of each of the enzymes was also measured. The results obtained with enzymes mutated at positions 14, 38, and 214 are in accordance with published data on Drosophila alcohol dehydrogenase and suggest interactions of these residues with the cofactor NAD. The introduction of a methyl group at residue Gly-19 abolished the ability of the enzyme to utilize NADP instead of NAD. This reflects a proximity of residue Gly-19 to the ribose ring of the bound cofactor. This result, coupled to the three-dimensional model built for Drosophila alcohol dehydrogenase, suggests a binding mechanism for the cofactor NAD different from that found for 3 alpha,20 beta-dehydroxysteroid dehydrogenase and similar to that found in the crystal structure of rat liver dihydropteridine reductase. The model of Drosophila alcohol dehydrogenase also enables many previous observations from chemical modification, sequence comparisons, site-directed mutagenesis, and limited proteolysis experiments to be placed into a structural context. An active site architecture is proposed involving a loop closure mechanism similar to that of lactate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The active site architecture of a short-chain dehydrogenase defined by site-directed mutagenesis and structure modeling. 800 69

Cod liver alcohol dehydrogenase of class-hybrid properties has been crystallized as an NAD(+)-pyrazole complex in the monoclinic space group P2(1) with cell dimensions a = 103.3 A, b = 47.4 A, c = 80.7 A, beta = 104.6 degrees, and with one dimer in the asymmetric unit. The position of the dimer molecule in the crystal was determined by molecular replacement methods at 3.0 A resolution. The successful search model was the poly-alanine structure of the horse enzyme. Side chains were then replaced according to the amino acid sequence of the cod enzyme, and the structure has been refined at 2.8 A to an R-factor of 0.26. Cod liver class III alcohol dehydrogenase crystallizes in the monoclinic space group C2 with cell dimensions a = 127.5 A, b = 76.6 A, c = 93.4 A, beta = 99.4 degrees and with probably one dimer in the asymmetric unit.
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PMID:Crystallisation and crystallographic investigations of cod alcohol dehydrogenase class I and class III enzymes. 806 9

2-Chloroacetaldehyde (CAA)-induced cytotoxicity in isolated hepatocytes was enhanced markedly if hepatocyte alcohol or aldehyde dehydrogenase was inhibited prior to CAA addition. Hepatocyte GSH depletion, ATP depletion and lipid peroxidation by CAA were also enhanced markedly. Furthermore, CAA was about 10- and 70-fold more cytotoxic than its oxidative or reductive metabolite chloroacetate or chloroethanol, respectively. Nutrients such as lactate, xylitol, sorbitol or glycerol, which increase cytosolic NADH levels, prevented CAA cytotoxicity in normal hepatocytes but further enhanced cytotoxicity toward alcohol dehydrogenase inactivated hepatocytes, suggesting that increased cytosolic NADH reduces CAA via alcohol dehydrogenase in normal hepatocytes but prevents CAA oxidation in alcohol dehydrogenase inactivated hepatocytes. However, increasing cytosolic NADH levels with ethanol or NADH-generating nutrients after CAA had been metabolized also prevented cytotoxicity and caused a partial ATP recovery, whereas oxidation of cytosolic NADH with pyruvate markedly increased cytotoxicity. This indicates that cytotoxic CAA concentrations cause oxidative stress and that ATP levels can be restored if cellular redox homeostasis is normalized with reductants. Furthermore, except for fructose, nutrients that did not increase NADH did not affect CAA-induced cytotoxicity. Fructose also caused a partial ATP recovery, and its protection was prevented by the glycolytic inhibitor fluoride. Hepatocytes isolated from fasted animals were 4- to 6-fold more susceptible to CAA-induced ATP depletion and cytotoxicity. No lipid peroxidation occurred at these lower CAA concentrations. Furthermore, all nutrients, including alanine, glutamine and glucose, prevented cytotoxicity toward hepatocytes isolated from fasted animals. The susceptibility of hepatocytes to CAA cytotoxicity, therefore, depends on both cellular redox homeostasis and cellular energy supply.
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PMID:Chloroacetaldehyde-induced hepatocyte cytotoxicity. Mechanisms for cytoprotection. 809 90

The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; approximately 68% residue identities) than to the other classes (II, III, and V; approximately 60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kcat values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I.
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PMID:Mammalian class IV alcohol dehydrogenase (stomach alcohol dehydrogenase): structure, origin, and correlation with enzymology. 812 1

Histidinol dehydrogenase (HDH) catalyzes two sequential oxidation reactions to produce histidine from histidinol via histidinaldehyde. In HDH proteins so far reported, two Cys residues are conserved. From the results of the studies on Salmonella typhimurium HDH, it has been proposed that one of these two conserved Cys residues is involved in the thiohemiacetal formation at the aldehyde oxidation step [Grubmeyer and Gray (1986) Biochemistry 25, 4778-4784]. To clarify the reaction mechanism, we investigated the role of the conserved Cys residues by site-directed mutagenesis in cabbage HDH. Thus, Cys-112, that corresponds to the catalytic Cys residue of the Salmonella enzyme, and the other conserved one, Cys-149, were replaced with Ala, Ser, or Phe. All the Cys-112 mutant HDHs catalyzed both the alcohol dehydrogenase and aldehyde dehydrogenase reactions, producing 1 mol of L-histidine during the reduction of 2 mol of NAD+, as did the wild type HDH. Site-directed mutagenesis at Cys-149 did not cause significant changes in the catalytic properties, either. These observations, together with the results of detailed comparison of the catalytic properties of mutant HDHs, clearly indicate that neither Cys-112 nor Cys-149 is involved in the reaction, and ruled out the involvement of thiohemiacetal formation in the histidinol dehydrogenase reaction.
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PMID:Site-directed mutagenesis shows that the conserved cysteine residues of histidinol dehydrogenase are not essential for catalysis. 813 43

The homodimeric alcohol dehydrogenase gene produce of maize (Zea mays L.) Adh1-1S1108 mutation was purified and compared with the parental Adh1-1S enzyme. The mutant alcohol dehydrogenase activity had pH optima and substrate specificity similar to those of the parental enzyme, but exhibited somewhat increased and decreased Km values for acetaldehyde and NADH, respectively. The mutant enzyme was also markedly less stable than the enzyme from parental tissues to temperatures as low as 50 degrees C. Sequence analysis of a polymerase chain reaction (PCR)-generated cDNA clone revealed a G-to-C mutation at position 406 and a C-to-T mutation at position 974. These would result in residue 103 of each protein subunit being changed from an alanine to a proline and residue 292 being changed from an alanine to a valine. Whether one or both of these changes in primary sequence is responsible for the altered substrate affinities and stability is not yet understood.
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PMID:Characterization of mutation-induced changes in the maize (Zea mays L.) ADH1-1S1108 alcohol dehydrogenase. 816 23

The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity.
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PMID:Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation. 846 Jan 64

The relationship between the size of the substrate binding pocket and the catalytic reactivities with varied alcohols was studied with the Saccharomyces cerevisiae alcohol dehydrogenase I (ScADH) and compared with the liver enzymes from horse (EqADH, EE isoenzyme) and monkey (MmADH alpha, alpha-isoenzyme). The yeast enzyme is most active with ethanol, and its activity decreases as the size of the alcohol is increased, whereas the activities of the liver enzymes increase with larger alcohols. The substrate pocket in ScADH was enlarged by single substitutions of Thr-48 to Ser (T48S), Trp-57 to Met (W57M), and Trp-93 to Ala (W93A), and a double change, T48S:W93A, and a triple, T48S:W57M:W93A. The T48S enzyme has the same pattern of activity (V/K) as wild-type ScADH for linear primary alcohols. The W57M enzymes have lowered reactivity with primary and secondary alcohols. The W93A and T48S:W93A enzymes resemble MmADH alpha in having an inverted specificity pattern for primary alcohols, being 3- and 10-fold more active on hexanol and 350- and 540-fold less active on ethanol, and are as reactive as the liver enzymes with long chain primary alcohols. The three Ala-93 enzymes also acquired weak activity on branched chain alcohols and cyclohexanol.
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PMID:Inversion of the substrate specificity of yeast alcohol dehydrogenase. 846 7


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