Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide

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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...

Section: Resultsmentioning

confidence: 93%

Kinetic Cooperativity of Human Liver Alcohol Dehydrogenase γ2

Charlier

Plapp

2000

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“…Reagents in the U.S.E. mutagenesis kit (Amersham Pharmacia Biotech) and mutagenesis primers: ACGTGTGTGTGCC(A/C/G)TACTGA-CATC(A/C)ATGCCACCGATCC and 5Ј-GATGTGGGTTCTCAACCG-GCTACGGGGCTG-3Ј were used to alter the codons and subsequently replacing Pro 47 for His, Asn 51 for His, and Ser 182 for Thr (nucleotides corresponding to the changed codons are underlined). Selection was based on the elimination of the unique XbaI site of the pET29 vector according to the method described by Deng and Nickoloff (24).…”

Section: Methodsmentioning

confidence: 99%

A Novel Subtype of Class II Alcohol Dehydrogenase in Rodents

Svensson¹,

Strömberg²,

Höög

1999

Mice and rats were found to possess class II alcohol dehydrogenases with novel enzymatic and structural properties. A cDNA was isolated from mouse liver and the encoded alcohol dehydrogenase showed high identity (93.1%) with the rat class II alcohol dehydrogenase which stands in contrast to the pronounced overall variability of the class II line. The two heterologously expressed rodent class II enzymes exhibited over 100-fold lower catalytic efficiency (k cat /K m ) for oxidation of alcohols as compared with other alcohol dehydrogenases and were not saturated with ethanol. Hydride transfer limited the rate of octanol oxidation as indicated by a deuterium isotope effect of 4.8. The mutation P47H improved hydride transfer and turnover rates were increased to the same level as for the human class II enzyme. Michaelis constants for alcohols and aldehydes were decreased while they were increased for the coenzyme. The rodent class II enzymes catalyzed reduction of p-benzoquinone with about the same maximal turnover as for the human form. This activity was not affected by the P47H mutation while a S182T mutation increased the K m value for benzoquinone 10-fold. -Hydroxy fatty acids were catalyzed extremely slow but functioned as potent inhibitors by binding to the enzyme-NAD ؉ complex. All these data indicate that the mammalian class II alcohol dehydrogenase line is divided into two structurally and functionally distinct subgroups.

“…Further, a high K i value for 2-nonanone in product inhibition studies (Table II) and an unmeasurable K d in equilibrium binding studies hint for a low affinity of this product for AOR. Horse LADH demonstrates an ordered bi bi kinetic mechanism with cofactor binding first (27,28). It is possible that other AOR substrates, with greater product affinities, may be metabolized according to this mechanism.…”

Section: Deuterium and Solvent Kinetic Isotope Effects-both [4s-2 H] mentioning

confidence: 99%

The Catalytic and Kinetic Mechanisms of NADPH-dependent Alkenal/one Oxidoreductase

Dick

Kensler

2004

NADPH-dependent alkenal/one oxidoreductase (AOR) from the rat is a phase 2/antioxidative enzyme that is known to catalyze the reduction of the carbon-carbon double bond of ␣,␤-unsaturated aldehydes and ketones. It is also known for its leukotriene B 4 12-hydroxydehydrogenase activity. In order to begin to understand these dual catalytic activities and validate its classification as a reductase of the medium-chain dehydrogenase/ reductase family, an investigation of the mechanism of its NADPH-dependent activity was undertaken. Recombinant AOR and a 3-nonen-2-one substrate were used to perform steady-state initial velocity, product inhibition, and dead end inhibition experiments, which elucidated an ordered Theorell-Chance kinetic mechanism with NADPH binding first and NADP ؉ leaving last. A nearly 20-fold preference for NADPH over NADH was also observed. The dependence of kinetic parameters V and V/K on pH suggests the involvement of a general acid with a pK of 9.2. NADPH isomers stereospecifically labeled with deuterium at the 4-position were used to determine that AOR catalyzes the transfer of the pro-R hydride to the ␤-carbon of an ␣,␤-unsaturated ketone, illudin M. Two-dimensional nuclear Overhauser effect NMR spectra demonstrate that this atom becomes the R-hydrogen at this position on the metabolite. Using [4R-2 H]NADPH, small primary kinetic isotope effects of 1.16 and 1.73 for V and V/K, respectively, were observed and suggest that hydride transfer is not rate-limiting. Atomic absorption spectroscopy indicated an absence of Zn 2؉ from active preparations of AOR. Thus, AOR fits predictions made for medium-chain reductases and bears similar characteristics to well known medium-chain alcohol dehydrogenases. NAD(P)H-dependent alkenal/one oxidoreductase (AOR)1 is an enzyme that reduces the carbon-carbon double bond of a variety of ␣,␤-unsaturated aldehydes and ketones (1). It is coregulated in the rat with a variety of phase 2/antioxidative enzymes, including NAD(P)H:quinone reductase, glutathione S-transferases, and UDP-glucuronosyltransferases, through the Keap1/Nrf2 signaling pathway (2). ␣,␤-Unsaturated aldehydes and ketones are electrophilic and capable of reacting, via a Michael addition mechanism, with important cellular nucleophiles, which in turn leads to macromolecular (protein, DNA) dysfunction and cell death. Because saturated carbonyls lack this reactive moiety, they are often far less toxic. Thus, hydrogenation of the ␣,␤-double bond by AOR conceptually results in detoxication (1, 3).Of the AOR substrates identified, several are common environmental pollutants (methyl vinyl ketone and acrolein) or products of lipid peroxidation (4, 5). The latter process involves reaction of oxygen free radicals with polyunsaturated fatty acids to form aliphatic ␣,␤-unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE), 2-hexenal, and 2,4-decadienal (5). These reactive molecules probably mediate many of the detrimental effects of oxidative stress. 4HNE is extremely cytotoxic, an abundant product of lipid perox...