Rate constants for a mechanism including intermediates in the interconversion of ternary complexes by horse liver alcohol dehydrogenase

Sekhar, V. Chandra; Plapp, Bryce V.

doi:10.1021/bi00470a005

Cited by 112 publications

(165 citation statements)

References 45 publications

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“…This and previous studies of nicotinamide dehydrogenases (10,12,(22)(23)(24)(25)(26) have established the importance, to the free energy of activation, of a substituent adjacent to the face of the NAD ϩ or NADH nicotinamide ring and distal to the substrate. It has now been shown with HLADH⅐NAD ϩ ⅐PhCH 2 O Ϫ that anticorrelated motions of the V292 of the cofactor-binding domain, in particular, and V203 with motions of the substrate push cofactor C4 and substrate H-C7 into position to create reactive groundstate conformations (NACs).…”

Section: Discussionsupporting

confidence: 63%

“…It is generally appreciated that AM1-calculated values of energy barriers overestimate the barrier heights in comparison with ab initio and density functional results (21). By inclusion of the quantum-mechanical corrections to the vibration energy (21) (Ϸ2.8 kcal͞mol) to the calculated hydride-transfer barrier height, our calculated energy value (15.6 kcal͞mol) may be compared to the experimentally simulated tunneling value of Ϸ16 kcal͞mol (22,23).…”

Section: Resultsmentioning

confidence: 76%

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Anticorrelated motions as a driving force in enzyme catalysis: The dehydrogenase reaction

Luo

Bruice

2004

Proc. Natl. Acad. Sci. U.S.A.

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A present issue concerns the occurrence of rate-promoting vibrations, which assist in lowering barrier height in enzyme catalysis (1-5). A related issue addressed here is the question of whether certain thermal Brownian motions of an enzymesubstrate complex (E⅐S) participate in lowering the energy barrier of activation (6) by creating the most reactive groundstate conformations [near-attack conformers (NACs) (7)]. One such possibility is that ground-state conformations, which lead directly to the lowest energy-transition state (NACs), are formed with assistance of anticorrelated motions of residues proximal to the active site.From Eq. 1, the free energy of activation is equal to the sum of the standard free energies for formation of reactive E⅐NAC conformers (⌬GЊ NAC ) plus the activation energy (⌬G TS ‡ ) for conversion of E⅐NAC to E⅐TS as shown in Eq. 2.We report here the findings of a study of correlated and anticorrelated motions of an E⅐S complex, the role of certain anticorrelated motions in the formation of E⅐NAC, as well as the demonstration that reactions that pass through E⅐NAC follow a kinetically favored trajectory. The reaction of interest is the oxidation of a primary alcohol by horse liver alcohol dehydrogenase (HLADH⅐NAD ϩ ). The computational tools used have been molecular dynamics (MD) simulations, crosscorrelation analysis, and quantum mechanics (QM)͞molecular mechanics (MM).HLADH (EC 1.1.1.1) (8) consists of two subunits of identical composition in which a 12-stranded ␤-sheet makes up the central core (9). Each subunit binds a molecule of NAD ϩ and a Zn 2ϩ at the active site and an additional Zn 2ϩ , which is structural. Alcohol oxidation by NAD ϩ involves first the proton dissociation of RCH 2 OH followed by transfer of a hydride equivalent from RCH 2 O Ϫ to NAD ϩ (Eq. 3).In an earlier study we carried out MD simulations (10) for 1-2 ns the structures of HLADH⅐NAD ϩ ⅐PhCH 2 OH, HLADH⅐NAD ϩ ⅐PhCH 2 O Ϫ , and HLADH⅐NADH⅐PhCHO using one subunit covered by a 32-Å-radius pool of transferable intermolecular potential three-point water molecules. By doing so we were able to derive an ensemble of dynamic conformations for the reactive HLADH⅐NAD ϩ ⅐PhCH 2 O Ϫ species, define the paths of H ϩ transfer to water, and established the presence of a water channel, which facilitates proton shuttling (10). We reported that the Michaels complex assumed the conformation of a reactive conformer [NAC (7)] 60% of the time. We defined the NAC as a conformer in which the distances between the transferring of an H Ϫ equivalent from C7 of PhCH 2 O Ϫ and acceptor C4 of NAD ϩ are Յ3.0 Å (3.9 Å for the heavy atoms C7 and C4) and the angles of C7-H-NC4 range from 132°to 180°. Concurrently, an investigation from Karplus and coworkers (11) appeared in which the kinetic features, the adiabatic potential barrier heights, the rate constants (including quantum mechanical effects such as tunneling), and potential of mean force of the HLADH⅐NAD ϩ ⅐PhCH 2 O Ϫ species were described by use of self-consistent charge density functio...

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Section: Discussionsupporting

confidence: 63%

Section: Resultsmentioning

confidence: 76%

Anticorrelated motions as a driving force in enzyme catalysis: The dehydrogenase reaction

Luo

Bruice

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Many of the rate constants for the mechanism in Fig. 4 can be estimated from kinetic constants that have been determined for liver alcohol dehydrogenases (31,32). Since ADH is typically saturated with substrates during ethanol metabolism, the turnover number (V max ) of ADH controls the rate of ethanol metabolism, and this is usually controlled by the rate-limiting release of NADH, k ϩ4 , which is the only sensitive rate constant for the ADH reaction.…”

Section: Table I Inhibition Of Purified Liver Alcohol Dehydrogenases mentioning

confidence: 99%

Formamides Mimic Aldehydes and Inhibit Liver Alcohol Dehydrogenases and Ethanol Metabolism

Venkataramaiah

Plapp

2003

Journal of Biological Chemistry

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Formamides are unreactive analogues of the aldehyde substrates of alcohol dehydrogenases and are useful for structure-function studies and for specific inhibition of alcohol metabolism. They bind to the enzyme-NADH complex and are uncompetitive inhibitors against varied concentrations of alcohol. Fourteen new branched chain and chiral formamides were prepared and tested as inhibitors of purified Class I liver alcohol dehydrogenases: horse (EqADH E), human (HsADH1C*2), and mouse (MmADH1). In general, larger, substituted formamides, such as N-1-ethylheptylformamide, are better inhibitors of HsADH1C*2 and MmADH1 than of EqADH, reflecting a few differences in amino acid residues that change the sizes of the active sites. In contrast, the linear, alkyl (n-propyl and n-butyl) formamides are better inhibitors of EqADH and MmADH1 than of HsADH1C*2, probably because water disrupts van der Waals interactions. These enzymes are also inhibited strongly by sulfoxides and 4-substituted pyrazoles. The structure of EqADH complexed with NADH and (R)-N-1-methylhexylformamide was determined by x-ray crystallography at 1.6 Å resolution. The structure resembles the expected Michaelis complex with NADH and aldehyde, and shows for the first time that the reduced nicotinamide ring of NADH is puckered, as predicted for the transition state for hydride transfer. Metabolism of ethanol in mice was inhibited by several formamides. The data were fitted with kinetic simulation to a mechanism that describes the non-linear progress curves and yields estimates of the in vivo inhibition constants and the rate constants for elimination of inhibitors. Some small formamides, such as N-isopropylformamide, may be useful inhibitors in vivo.Vertebrate alcohol dehydrogenases (EC 1.1.1.1, ADH) 1 are involved in the metabolism of relatively non-polar alcohols and aldehydes (1). The five different human enzymes have broad substrate specificity, oxidizing primary and secondary alcohols and reducing their corresponding carbonyl compounds. Specific inhibitors of these dehydrogenases would be useful for defining substrate specificity, for studying the physiological roles of the enzymes and for preventing the oxidation of methanol and ethylene glycol to toxic products (2-6).Formamides and sulfoxides are analogues of the carbonyl substrates, are bound preferentially to the enzyme-NADH complex, and are potent uncompetitive inhibitors against varied concentrations of alcohol (7-15). Uncompetitive inhibitors could be especially useful for controlling alcohol metabolism since they are effective even when the concentrations of alcohol are saturating. Previous studies identified some potent uncompetitive inhibitors for human (14, 15), horse (10,11,14,15), monkey (11), and rat (10, 11) liver alcohol dehydrogenases. We now extend the studies with 14 new branched-chain and chiral formamides in order to explore active site topologies and to make more effective inhibitors for the mouse (MmADH1) and human (HsADH1C*2) enzymes. For comparison, we also studied the potency of fi...

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“…The coenzyme NAD ÷ was lyophilised from distilled water. HLADH from Sigma (specific activity 1-2 U/mg) was used because its ethanol content, less than 0.15%, proved to be low enough not to disturb activity determinations using isopropanol as substrate, making extensive dialysis or gel filtration of the enzyme [22] superfluous. The PEG-NADH was oxidized to PEG-NAD ÷ as described by Biickmann et al [21].…”

Section: Methodsmentioning

confidence: 99%

Kinetic and modelling studies of NAD+ and poly(ethylene glycol)-bound NAD+ in horse liver alcohol dehydrogenase

Vanhommerig

Sluyterman

Meijer

1996

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Rate constants for a mechanism including intermediates in the interconversion of ternary complexes by horse liver alcohol dehydrogenase

Cited by 112 publications

References 45 publications

Anticorrelated motions as a driving force in enzyme catalysis: The dehydrogenase reaction

Anticorrelated motions as a driving force in enzyme catalysis: The dehydrogenase reaction

Formamides Mimic Aldehydes and Inhibit Liver Alcohol Dehydrogenases and Ethanol Metabolism

Kinetic and modelling studies of NAD+ and poly(ethylene glycol)-bound NAD+ in horse liver alcohol dehydrogenase

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