Alcohol dehydrogenase hydride transfer

Figure 1.9 Examples of functionally important intrinsic metal atoms in proteins, (a) The di-iron center of the enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The iron atoms are bridged by a glutamic acid residue and a negatively charged oxygen atom called a p-oxo bridge. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules, (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains. During catalysis zinc binds an alcohol molecule in a suitable position for hydride transfer to the coenzyme moiety, a nicotinamide, [(a) Adapted from P. Nordlund et al., Nature 345 593-598, 1990.)...

FIGURE 16.13 Liver alcohol dehydrogenase catalyzes the transfer of a hydride ion (H ) from NADH to acetaldehyde (CH3CHO), forming ethanol (CH3CH9OH). 

NAD (P) " -dependent enzymes are stereospecific. Malate dehydrogenase, for example, transfers a hydride to die pro-/ position of NADH, whereas glyceraldehyde-3-phosphate dehydrogenase transfers a hydride to die pro-5 position of the nicotinamide. Alcohol dehydrogenase removes a hydride from the pro-i position of edianol and transfers it to die pro-i position of NADH. 

Stone CL, Bosron WF, Dunn MF. Amino acid substitutions at position 47 of human beta 1 beta 1 and beta 2 beta 2 alcohol dehydrogenases affect hydride transfer and coenzyme dissociation rate constants. J Biol Chem 1993 268 892-899... 

When the enzyme alcohol dehydrogenase converts acetaldehyde to ethanol, NADH acts as a reducing agent by transferring a hydride from C4 of the nicotinamide ring to the carbonyl group of acetaldehyde. 

Rhin(bpy)3]3+ and its derivatives are able to reduce selectively NAD+ to 1,4-NADH in aqueous buffer.48-50 It is likely that a rhodium-hydride intermediate, e.g., [Rhni(bpy)2(H20)(H)]2+, acts as a hydride transfer agent in this catalytic process. This system has been coupled internally to the enzymatic reduction of carbonyl compounds using an alcohol dehydrogenase (HLADH) as an NADH-dependent enzyme (Scheme 4). The [Rhin(bpy)3]3+ derivative containing 2,2 -bipyridine-5-sulfonic acid as ligand gave the best results in terms of turnover number (46 turnovers for the metal catalyst, 101 for the cofactor), but was handicapped by slow reaction kinetics, with a maximum of five turnovers per day.50... 

Modelling the effects of Ser-48 in the hydride transfer step of liver alcohol dehydrogenase,... 

Since many of the transformations undergone by metabolites involve changes in oxidation state, it is understandable that cofactors have been developed to act as electron acceptors/ donors. One of the most important is that based on NAD/NADP. NAD+ can accept what is essentially two electrons and a proton (a hydride ion) from a substrate such as ethanol in a reaction catalysed by alcohol dehydrogenase, to give the oxidized product, acetaldehyde and the reduced cofactor NADH plus a proton (Figure 5.2). Whereas redox reactions on metal centres usually involve only electron transfers, many oxidation/reduction reactions in intermediary metabolism, as in the case above, involve not only electron transfer but... 

Alcohol dehydrogenases are a class of zinc enzymes, which catalyse the oxidation of primary and secondary alcohols to the corresponding aldehyde or ketone by the transfer of a hydride anion to NAD+ with release of a proton ... 

Thus, the role of zinc in the dehydrogenation reaction is to promote deprotonation of the alcohol, thereby enhancing hydride transfer from the zinc alkoxide intermediate. Conversely, in the reverse hydrogenation reaction, its role is to enhance the electrophilicity of the carbonyl carbon atom. Alcohol dehydrogenases are exquisitely stereo specific and by binding their substrate via a three-point attachment site (Figure 12.7), they can distinguish between the two-methylene protons of the prochiral ethanol molecule. 

Probably the most extensively studied enzymes are those from alcohol dehydrogenase family. One enzyme from this series which has been thoroughly examined both experimentally and theoretically is liver alcohol dehydrogenase (LADH). It catalyzes the reversible conversion of an alcohol to an aldehyde by transferring hydride from substrate to the cofactor (NAD+) ... 

In the following year, Cleland and his coworkers reported further and more emphatic examples of the phenomenon of exaltation of the a-secondary isotope effects in enzymic hydride-transfer reactions. The cases shown in Table 1 for their studies of yeast alcohol dehydrogenase and horse-liver alcohol dehydrogenase would have been expected on traditional grounds to show kinetic isotope effects between 1.00 and 1.13 but in fact values of 1.38 and 1.50 were found. Even more impressively, the oxidation of formate by NAD was expected to exhibit an isotope effect between 1.00 and 1/1.13 = 0.89 - an inverse isotope effect because NAD" was being converted to NADH. The observed value was 1.22, normal rather than inverse. Again the model of coupled motion, with a citation to Kurz and Frieden, was invoked to interpret the findings. 

Horse liver alcohol dehydrogenase and the F93W mutant, hydride transfer from henzyl alcohol to NAD in MeOH/water. 

Horse liver alcohol dehydrogenase, F93W mutant with 1224 also mutated to G,A,V,L. hydride transfer from benzyl alcohol to NAD Heterotetrameric sarcosine oxidase of Arthrobacter sp. 1-IN, proton transfer from adduct of FAD with sarcosine-(CH3) and sarcosine-(CD3)... 

Isotope effects have also been applied extensively to studies of NAD+/NADP+-linked dehydrogenases. We typically treat these enzymes as systems whose catalytic rates are limited by product release. Nonetheless, Palm clearly demonstrated a primary tritium kinetic isotope effect on lactate dehydrogenase catalysis, a finding that indicated that the hydride transfer step is rate-contributing. Plapp s laboratory later demonstrated that liver alcohol dehydrogenase has an intrinsic /ch//cd isotope effect of 5.2 with ethanol and an intrinsic /ch//cd isotope effect of 3-6-4.3 with benzyl alcohol. Moreover, Klin-man reported the following intrinsic isotope effects in the reduction of p-substituted benzaldehydes by yeast alcohol dehydrogenase kn/ko for p-Br-benzaldehyde = 3.5 kulki) for p-Cl-benzaldehyde = 3.3 kulk for p-H-benzaldehyde = 3.0 kulk for p-CHs-benzaldehyde = 5.4 and kn/ko for p-CHsO-benzaldehyde = 3.4. 

Alcohol dehydrogenases (ADH EC 1.1.1.1), for which several X-ray structures are available ", catalyze the biological oxidation of primary and secondary alcohols via the formal transfer of a hydride anion to the oxidized form of nicotinamide adenine dinucleotide (NAD ), coupled with the release of a proton. Liver alcohol dehydrogenase (LADH) consists of two similar subunits, each of which contains two zinc sites, but only one site within each subunit is catalytically active. The catalytic zinc is coordinated in a distorted tetrahedral manner to a histidine residue, two cysteine residues and a water molecule. The remaining zinc is coordinated tetrahedrally to four cysteine residues and plays only a structural role . 

A Zn2+ at the active site polarizes the carbonyl oxygen of acetaldehyde, allowing transfer of a hydride ion (red) from the reduced cofactor NADH. The reduced intermediate acquires a proton from the medium (blue) to form ethanol. Alcohol Dehydrogenase Mechanism... 

One step or two-step transfer Another major question about dehydrogenases is whether the hydrogen atom that is transferred moves as a hydride ion, as is generally accepted, or as a hydrogen atom with separate transfer of an electron and with an intermediate NAD or NADPH free radical. In one study para-substituted benzaldehydes were reduced with NADH and NAD2H using yeast alcohol dehydrogenase as a catalyst.30 This permitted the application of the Hammett equation (Box 6-C) to the rate data. For a series of benzaldehydes for which o+ varied widely, a value... 

The chemistry of flavins is complex, a fact that is reflected in the uncertainity that has accompanied efforts to understand mechanisms. For flavoproteins at least four mechanistic possibilities must be considered.1533 233 (a) A reasonable hydride-transfer mechanism can be written for flavoprotein dehydrogenases (Eq. 15-23). The hydride ion is donated at N-5 and a proton is accepted at N-l. The oxidation of alcohols, amines, ketones, and reduced pyridine nucleotides can all be visualized in this way. Support for such a mechanism came from study of the nonenzymatic oxidation of NADH by flavins, a reaction that occurs at moderate speed in water at room temperature. A variety of flavins and dihydropyridine derivatives have been studied, and the electronic effects observed for the reaction are compatible with the hydride ion mecha-nism.234 236... 

The NAD+-dependent alcohol dehydrogenase from horse liver contains one catalytically essential zinc ion at each of its two active sites. An essential feature of the enzymic catalysis appears to involve direct coordination of the enzyme-bound zinc by the carbonyl and hydroxyl groups of the aldehyde and alcohol substrates. Polarization of the carbonyl group by the metal ion should assist nucleophilic attack by hydride ion. A number of studies have confirmed this view. Zinc(II) catalyzes the reduction of l,10-phenanthroline-2-carbaldehyde by lV-propyl-l,4-dihy-dronicotinamide in acetonitrile,526 and provides an interesting model reaction for alcohol dehydrogenase (Scheme 45). The model reaction proceeds by direct hydrogen transfer and is absolutely dependent on the presence of zinc(II). The zinc(II) ion also catalyzes the reduction of 2- and 4-pyridinecarbaldehyde by Et4N BH4-.526 The zinc complex of the 2-aldehyde is reduced at least 7 x 105 times faster than the free aldehyde, whereas the zinc complex of the 4-aldehyde is reduced only 102 times faster than the free aldehyde. A direct interaction of zinc(II) with the carbonyl function is clearly required for marked catalytic effects to be observed. 

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