Hydride transfer reactions, NADH reaction mechanism

Finally it was reasoned that if the mechanism of Eq. (4.11a) involved an electron transfer or a hydrogen-atom transfer from the NADH analog to the acridinium species, then such processes should surely occur in the system of Eq. (4.11b). The fact that this did not occur, in spite of the electron transfer being thermodynamically favorable, demonstrated the extreme propensity of the NADH/flavin system for hydride-transfer reaction. 

Linear correlations between hydride transfer reactions of NADH analogues with (L)FeIV(0)]2 f and CI4Q in Figure 2.14 imply that the hydride transfer mechanism of (L)FeIV(0)]2+ is virtually the same as that of C14Q." Although there is still debate on the mechanism(s) of hydride transfer from NADH analogues to hydride acceptors in terms of an ET pathway versus a one-step hydride transfer pathway, the ET pathway is now well accepted for hydride transfer from NADH analogues to hydride acceptors... 

FIGURE 19-9 IMADH ubiquinone oxidoreductase (Complex I). Complex I catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the iron-sulfur protein N-2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses into the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons. The detailed mechanism that couples electron and proton transfer in Complex I is not yet known, but probably involves a Q cycle similar to that in Complex III in which QH2 participates twice per electron pair (see Fig. 19-12). Proton flux produces an electrochemical potential across the inner mitochondrial membrane (N side negative, P side positive), which conserves some of the energy released by the electron-transfer reactions. This electrochemical potential drives ATP synthesis. 

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 enzyme-product complexes of the yeast enzyme dissociate rapidly so that the chemical steps are rate-determining.31 This permits the measurement of kinetic isotope effects on the chemical steps of this reaction from the steady state kinetics. It is found that the oxidation of deuterated alcohols RCD2OH and the reduction of benzaldehydes by deuterated NADH (i.e., NADD) are significantly slower than the reactions with the normal isotope (kn/kD = 3 to 5).21,31 This shows that hydride (or deuteride) transfer occurs in the rate-determining step of the reaction. The rate constants of the hydride transfer steps for the horse liver enzyme have been measured from pre-steady state kinetics and found to give the same isotope effects.32,33 Kinetic and kinetic isotope effect data are reviewed in reference 34 and the effects of quantum mechanical tunneling in reference 35. 

ADH features another catalytic triad, Ser-Tyr-Lys. Whereas the liver ADH kinetic mechanism is highly ordered, coenzyme associating first and dissociating last, the yeast ADH mechanism is largely random. In both cases, the actual chemical reaction is a hydride transfer. In the oxidation of secondary alcohols by Drosophila ADH (DADH), the release of NADH from the enzyme-NADH complex is the rate-limiting step, so vmax is independent of the chemical nature of the alcohol. With primary alcohols, as vmax is much lower and depends on the nature of alcohol, Theorell-Chance kinetics are not observed and the rate-limiting step is the chemical interconversion from alcohol to aldehyde. 

To compare these two mechanisms, an NADH model without the recognition site was synthesised. The contribution of the flavin binding to the rate constant was thus evaluated and it was shown that the proximity of flavin and NADH model influenced the electron transfer rate. Mechanistic computations helped to show that with the appropriate NADH model system, both components were optimally arranged for the electron transfer. Although the exact mechanism of the reaction is still under debate, the kinetic isotope effect experiment indicated that in this case, the hydrogen at 4-position was transferred in the rate determining step which supported the hydride mechanism. 

FabI catalyzes the NADH- or NADPH-dependent reduction of 0 ,/3-unsaturated enoyl-ACPs in which the proAS hydrogen of the cofactor is transferred as a hydride to the C3 carbon of the substrate. Both hydride transfer and protonation occur on the same face of the double bond sytr. si face at C3 and re face at C2), yielding a product in which the 2R and 3S hydrogens are added during the reaction. Mechanistic studies on the FabI enzyme from M. tuberculosis (InhA) are consistent with a stepwise mechanism, in which hydride transfer generates an enolate intermediate that is subsequently protonated to generate the product. ... 

Fig. 4 Reaction mechanism of dTDP-glucose 4,6-dehydratase as proposed by Allard et al. [125], The first half of the reaction involves abstraction of a proton from the 4 hydroxyl group, and hydride transfer from C-4 to NAD. In the next step, a proton is removed from the C-5 atom of the sugar, and the C-6 hydroxyl group is protonated and eliminated as water. This yields the 4-keto-5,6-ene intermediate. The product is obtained after hydride transfer from NADH to carbon atom C-6 and protonation of carbon atom C-5...

An extensive investigation of the reaction mechanism and kinetics between NADH and a series of ferrocenium hex-afluorophosphate derivatives was made by Miller and coworkers [129, 130]. This investigation was pursued because the reaction between NADH and one-electron no-proton acceptors is abnormal in comparison with most biological reactions with NADH where a net transfer to the oxidizing agent of a hydride equivalent from... 

The mechanism of this oxidation for the enzyme liver alcohol dehydrogenase is shown for the reaction of 83, where ethanol is bound to the active site of the enzyme to give 84 via proton abstraction and then hydride transfer to generate acetaldehyde (see 85). NAD+ binds to the active site of the enzyme to induce a conformational change (see Chapter 8 for conformation) to close the active site. The oxidation of ethanol to acetaldehyde (ethanal) is accompanied by reduction of NAD+ to NADH, as shown in the illustration. 

A carbon-based hydride reducing reagent in biological systems is nicotinamide adenine dinucleotide, NADH, which reduces carbonyl compounds by a mechanism related to the Cannizzaro reaction. In these reactions, NADH transfers a hydride to a carbonyl compound to yield an alcohol and NAD, as shown. 

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