Reactivity of horse liver alcohol dehydrogenase with 3-methylcyclohexanols

Lee, Kang Man; Dahlhauser, Keith F.; Plapp, Bryce V.

doi:10.1021/bi00409a060

Cited by 19 publications

(11 citation statements)

References 22 publications

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“…1) were simulated using determined or estimated values for the rate constants (Table III) for each step of the abortive complex pathway in Scheme 1. The values for constants k 2 , k 3 , k Ϫ3, k 4 , and k Ϫ4 were taken from studies with horse liver enzyme for each of the respective alcohols (35,36). Rate constants k 1 , k 5 , k Ϫ5 , k Ϫ6 /k 6 (for reactions with ethanol and cyclohexanol), and k Ϫ7 were measured by the experiments described above, and an estimate for k Ϫ1 was calculated from K ia (Table II) and the value measured for k 1 .…”

Section: Resultsmentioning

confidence: 99%

Kinetic Cooperativity of Human Liver Alcohol Dehydrogenase γ2

Charlier

Plapp

2000

Journal of Biological Chemistry

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Previous studies showed that natural human liver alcohol dehydrogenase ␥ exhibits negative cooperativity (substrate activation) with ethanol. Studies with the recombinant ␥ 2 isoenzyme now confirm that observation and show that the saturation kinetics with other alcohols are also nonhyperbolic, whereas the kinetics for reactions with NAD ؉ , NADH, and acetaldehyde are hyperbolic. The substrate activation with ethanol and 1-butanol are explained by an ordered mechanism with an abortive enzyme-NADH-alcohol complex that releases NADH more rapidly than does the enzyme-NADH complex. In contrast, high concentrations of cyclohexanol produce noncompetitive substrate inhibition against varied concentrations of NAD ؉ and decrease the maximum velocity to 25% of the value that is observed at optimal concentrations of cyclohexanol. Transient kinetics experiments show that cyclohexanol inhibition is due to a slower rate of dissociation of NADH from the abortive enzyme-NADH-cyclohexanol complex than from the enzyme-NADH complex. Fluorescence quenching experiments confirm that the alcohols bind to the enzyme-NADH complex. The nonhyperbolic saturation kinetics for oxidation of ethanol, cyclohexanol, and 1-butanol are quantitatively explained with the abortive complex mechanism. Physiologically relevant concentrations of ethanol would be oxidized predominantly by the abortive complex pathway.Liver alcohol dehydrogenases (E.C. 1.1.1.1) catalyze the reversible oxidation of alcohols using NAD ϩ as a cofactor. Class I alcohol dehydrogenases from human liver are homo-and heterodimers comprised of ␣, ␤, and ␥ subunits (1). HsADH␤ 1 and HsADH␥ have high catalytic efficiencies on ethanol and contribute significantly to its metabolism (1). Polymorphisms in the ADH3 gene lead to the isoenzymes HsADH␥ 1 , which has Gln-271 and Val-349, and HsADH␥ 2 , with Arg-271 and Ile-349 (3). Genotyping indicates the allele frequency for HsADH␥ 2 is about 10% in East Asians and 43% in Europeans (4, 5). The V max for ethanol oxidation by HsADH␥ 1 is 2.5 times higher than that for HsADH␥ 2 (1, 6), and it was suggested that susceptibility to alcoholism and cirrhosis may be associated with the presence of HsADH␥ 2 (1, 7). However, extensive studies have not established a correlation (4,5,8). Further insights into the possible roles of alcohol dehydrogenases in alcoholism require quantitative descriptions of the kinetics of the various enzymes involved, but the properties of HsADH␥ are a challenge because both isoenzymes exhibit negative cooperativity for ethanol oxidation (6), and the mechanism has not yet been described.The negative cooperativity could arise from different mechanisms (9). Subunit interactions or "half-of-the-sites" reactivity (an extreme case of negative cooperativity) were reported for the horse liver E (ethanol active) enzyme (10, 11) but were later disputed (12)(13)(14). Other studies have found nonadditivity in the heterodimers of horse liver enzymes (15) and human liver enzymes (16), suggesting that the constituent subunits do not act...

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Section: Resultsmentioning

confidence: 99%

Kinetic Cooperativity of Human Liver Alcohol Dehydrogenase γ2

Charlier

Plapp

2000

Journal of Biological Chemistry

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“…Horse liver ADH has a large substrate binding site and readily accommodates and efficiently oxidizes benzyl alcohol, cyclohexanol, and branched chain alcohols [39–41]. The horse enzyme has much higher activity than Sce ADH1 does on branched chain alcohols [4].…”

Section: Discussionmentioning

confidence: 99%

Activity of yeast alcohol dehydrogenases on benzyl alcohols and benzaldehydes

Pal

Park

Plapp

2009

Chemico-Biological Interactions

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The substrate specificities of yeast alcohol dehydrogenases I and II from Saccharomyces cerevisiae (SceADH1 and SceADH2) and Saccharomyces carlsbergensis (ScbADH1) were studied. For this work, the gene for the S. carlsbergensis ADH1 was cloned, sequenced and expressed. The amino acid sequence of ScbADH1 differs at four positions as compared to SceADH1, including substitutions of two glutamine residues with glutamic acid residues, and has the same sequence as the commercial yeast enzyme, which apparently is prepared from S. carlsbergensis. The electrophoretic mobilities of ScbADH1, SceADH2 and commercial ADH are similar. The kinetics and specificities of ScbADH1 and SceADH1 acting on branched, long-chain and benzyl alcohols are very similar, but the catalytic efficiency of SceADH2 is about 10 to 100-fold higher on these substrates. A three dimensional structure of SceADH1 shows that the substrate binding pocket has Met-270, whereas SceADH2 has Leu-270, which allows larger substrates to bind. The reduction of a series of p-substituted benzaldehydes catalyzed by SceADH2 is significantly enhanced by electronwithdrawing groups, whereas the oxidation of p-substituted aromatic alcohols may be only slightly affected by the substituents. The substituent effects on catalysis generally reflect the effects on the equilibrium constant for the reaction, where electron-withdrawing substituents favor alcohol. The results are consistent with a transition state that is electronically similar to the alcohol, supporting previous results obtained with commercial yeast ADH.

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“…Gordon et al, 1986;Cox & Boeker, 1987), FITSIM is designed to allow, at least in principle, the analysis of nearly any user-entered mechanism. Examples of the use of this program are given by Zimmerle et al (1987), Kurz et al (1987), Sekhar & Plapp (1988) and Lee et al (1988).…”

Section: Introductionmentioning

confidence: 99%

Analysis of progress curves by simulations generated by numerical integration

Zimmerle

Frieden

1989

Biochemical Journal

231

206

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A highly flexible computer program written in FORTRAN is presented which fits computer-generated simulations to experimental progress-curve data by an iterative non-linear weighted least-squares procedure. This fitting procedure allows kinetic rate constants to be determined from the experimental progress curves. Although the numerical integration of the rate equations by a previously described method [Barshop, Wrenn & Frieden (1983) Anal. Biochem. 130, 134-145] is used here to generate predicted curves, any routine capable of the integration of a set of differential equations can be used. The fitting program described is designed to be widely applicable, easy to learn and convenient to use. The use, behaviour and power of the program is explored by using simulated test data.

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Reactivity of horse liver alcohol dehydrogenase with 3-methylcyclohexanols

Cited by 19 publications

References 22 publications

Kinetic Cooperativity of Human Liver Alcohol Dehydrogenase γ2

Kinetic Cooperativity of Human Liver Alcohol Dehydrogenase γ2

Activity of yeast alcohol dehydrogenases on benzyl alcohols and benzaldehydes

Analysis of progress curves by simulations generated by numerical integration

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