Alternative pathways and reactions of benzyl alcohol and benzaldehyde with horse liver alcohol dehydrogenase

Shearer, Gretchen L.; Kim, Keehyuk; Lee, Kang Man; Wang, C. K.; Plapp, Bryce V.

doi:10.1021/bi00092a031

Cited by 82 publications

(89 citation statements)

References 65 publications

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“…The equilibrium in solution favors the alcohol-NAD þ side of the reaction (at catalytically relevant pH) (7), but the solution equilibrium is not directly relevant to the rate-limiting hydride transfer step under study. Measurements of internal (on the enzyme) equilibrium gave a value of K eq ¼ 0.15 in favor of the alcohol-NAD þ side, predicting a late TRS (9), but the internal equilibrium is close to unity and very likely a combination of at least two steps: the deprotonation of the alcohol and the hydride transfer that follows (46). Indeed, QM/MM calculations for this reaction found that the hydride transfer step is exothermic, despite the fact that the overall reaction was endothermic (24,27).…”

Section: Resultsmentioning

confidence: 99%

Elusive transition state of alcohol dehydrogenase unveiled

Roston

Kohen

2010

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

154

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For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (>1) 2°kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (<1), this KIE eludes the traditional interpretation of 2°KIEs. It does, however, enable the development of a comprehensive model for the "tunneling ready state" (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate.hydrogen tunneling | enzyme kinetic | secondary isotope effect | Swain-Schaad E nzymes enhance the rates of chemical reactions by many orders of magnitude, and extensive studies have uncovered many aspects of how they do so. The prevailing theory that enzymes stabilize the transition state (TS) relative to the reactants explains many phenomena, and a great deal of contemporary research focuses on how enzymes stabilize the TS. An unfortunate hindrance to developing an understanding of how enzymes stabilize the TS lies in the enigmatic nature of the TS. Many enzymatic hydrogen (proton, hydrogen, or hydride) transfers, for example, occur by quantum mechanical tunneling, where a particle passes through an energy barrier because of its wave properties (1-6).Yeast alcohol dehydrogenase (yADH) serves as an excellent model system for enzyme-catalyzed H transfer because unlike many other enzymes, the chemical step, oxidation of a primary alcohol to an aldehyde by NAD þ , is rate-limiting with aromatic substrates (7). Furthermore, the thermodynamics of the reaction allow for examination of both the forward (alcohol to aldehyde) and reverse (aldehyde to alcohol) reactions under similar conditions (8). This type of nicotinamide-dependent redox reaction appears ubiquitously in biology, and a detailed picture of the TS of such reactions may facilitate many medical and industrial applications.Intriguingly, decades of experiments on the reaction catalyzed by yADH have returned what appear to be contradictory results, some suggesting an early TS, whereas others point to a late TS (7-10). In pioneering studies, Klinman measured linear fr...

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Section: Resultsmentioning

confidence: 99%

Elusive transition state of alcohol dehydrogenase unveiled

Roston

Kohen

2010

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

154

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“…The aldehydes can also be reduced to the corresponding alcohols either by ADH or by aldehyde reductase. In addition, horse class I ADH has dismutase activity which results in both the corresponding alcohol and carboxylic acid from the aldehyde substrate [7,10,11]. The aim of the present study was to establish whether dismutation of aldehydes is catalysed also by human ADHs.…”

Section: Discussionmentioning

confidence: 99%

“…Kinetic constants indicate that class I Y2Y2 has a high catalytic efficiency for this reaction, while class I 91131 and class II have more moderate catalytic (Table 1). However, these constants may not be compared directly with those of aldehyde reduction, since kcatIKm are composed of different sets of fundamental rate constants [11]. The variation in efficiency of these ADHs reflects the structural differences in the substrate-binding pocket [21].…”

Section: Discussionmentioning

confidence: 99%

“…Reevaluation of this reaction has shown remarkably fast kinetics for the aliphatic aldehydes studied: acetaldehyde, butanal and octanal [10,11]. Simultaneous oxidation and reduction of aldehydes occur when horse liver ADH is incubated with aldehyde and NAD +.…”

Section: Introductionmentioning

confidence: 99%

“…1), and no net reduction of the coenzyme can be observed. In competition assays, the oxidation of aldehydes was as fast as the oxidation of the corresponding alcohols indicating a possible physiological role for ADH-catalysed aldehyde oxidation [11]. An important question is whether all ADHs possess this activity.…”

Section: Introductionmentioning

confidence: 99%

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Aldehyde dismutase activity of human liver alcohol dehydrogenase

et al. 1996

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Human alcohol dehydrogenases of class I and class II but not class III catalyse NAD+-dependent aldehyde oxidation in addition to the NADH-dependent aldehyde reduction. The two reactions are coupled, i.e. the enzymes display dismutase activity. Dismutase activity of recombinantly expressed human class I isozymes [~1[~1 and T2T2, class II and class III alcohol dehydrogenases was assayed with butanal as snbstrate by gas chromatographic-mass spectrometric quantitations of butanol and butyric acid. The class I Y2Y2 isozyme showed a pronounced dismutase activity with a high kcat, 1300 min -1, and a moderate Km, 1.2 mM. The class I ~1111 isozyme and the class II alcohol dehydrogenase showed moderate catalytic efficiencies for dismutase activity with lower kcat values, 60-75 min -1. 4-Methylpyrazole, a potent class I ADH inhibitor, inhibited the class I dismutation completely, but cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, did not affect the dismutation. The dismutase reaction might be important for metabolism of aldehydes during inhibition or lack of mitochondrial aldehyde dehydrogenase activity.

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Zn‐Dependent Medium‐Chain Dehydrogenases/Reductases

Meijers

Cedergren-Zeppezauer

2004

Handbook of Metalloproteins

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Medium‐chain dehydrogenases/reductases (MDRs) have about 350 to 390 amino acids and belong to a large superfamily of proteins including the Zn‐dependent alcohol dehydrogenases (ADHs) and several other Zn‐dependent activities. They are dimers or tetramers and utilize either NADH or NADPH as electron carriers referred to as cofactors. The oxidation/reduction process is a 2‐electron/1‐proton (hydride ion, H − ) transfer between substrate and cofactor. The compounds oxidized or reduced by these enzymes vary considerably in structure ranging from small primary and secondary alcohols/aldehydes/ketones (aliphatic and aromatic), sugars (sorbitol/fructose, glucose) to large substances like fatty acids, steroids, and retinoids. ADHs are widely distributed in nature and some MDR members are found in all species. An essential Zn ion at the active site is common to all MDRs but some members have two zinc ions per subunit. The 3D Structures of Zn‐dependent MDRs are highly similar and they essentially have the same polypeptide fold. The low sequence identity found sometimes could not immediately reveal this close structural relationship. Now numerous structures have been examined indicating that the evolutionary strategy for MDRs has been to vary amino acids preferably at the reaction center and further away in the substrate binding area. Thus, the size and shape of the substrate channel is adapted for small or large substrates, hydrophilic or hydrophobic substances, modifications attained via few mutations, or significant deletions/additions of amino acids. A further opportunity to create multiplicity has been to vary the control mechanism for the protein conformation change and the oligomeric state. Some enzymes undergo significant structural changes upon cofactor binding, while others do not. A transition state intermediate of NADH in complex with horse liver alcohol dehydrogenase, resolved to atomic resolution (≈1 Å), has revealed structural modifications of the cofactor associated with the activation process for hydride transfer. This involves an OH − pyridine ring adduct, connected to the zinc center. The distortion of the nicotinamide at the enzyme site, affected by the presence of the negatively charged oxygen ligand, influences three parameters: (i) the puckering of the ring, (ii) a deviation from a standard carbon–carbon double bond length, and (iii) accumulation of negative charge at the C4 atom promoting hydride transfer. The active site Zn‐center of this large enzyme group shows great flexibility with respect to metal–ligand geometry and dynamics. Structural and spectroscopic evidence suggest that Zn‐dependent MDRs should be included in the general family of Zn‐enzymes, which require water and hydroxide ion as reactants during catalysis.

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Alternative pathways and reactions of benzyl alcohol and benzaldehyde with horse liver alcohol dehydrogenase

Cited by 82 publications

References 65 publications

Elusive transition state of alcohol dehydrogenase unveiled

Elusive transition state of alcohol dehydrogenase unveiled

Aldehyde dismutase activity of human liver alcohol dehydrogenase

Zn‐Dependent Medium‐Chain Dehydrogenases/Reductases

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