Regeneration of NAD

A Regeneration of NAD that was converted to NADH by electron transfer during glycolysis must occur in order for glycolysis to continue. 

The mechanisms that maintain balance between NAD usage and regeneration differ under aerobic versus anaerobic conditions in tissues. 

In cells that are unable to transfer electrons to oxygen due to lack of mitochondria, eg, RBCs, or in vigorously exercising muscle cells (anaerobic conditions), NAD is regenerated by further metabolism of pyruvate. 

This reaction is reversible, and lactate can subsequently serve as an important source of carbons for gluconeogenesis in the liver. 

RBCs lack mitochondria and therefore depend on anaerobic glycolysis for energy needs. 

---

The electrocatalytic activity of novel redox films in regeneration of NAD/NADH has been investigated by means of chronoamperometry, hydrodynamic and potentiodynamic methods. In order to achieve the most efficient electrocatalytic properties indicated as both the highest heterogeneous rate constant and maximum sensitivity, the further optimization of electropolymerisation conditions has been made. 

Organometallics such as rhodium complex were also used for electrochemical regeneration of NAD(P)H from electrode (Figure 8.10) [7bj. 

Nonenzymatic regeneration of NAD(P)H requires the regioselective transfer of two electrons and a proton to NAD(P)+. Various rhodium(III) complexes are effective electrocatalysts capable of mimicking hydrogenase enzymes.48-54... 

FIGURE 12.10 Oxidation of methanol to C02, catalyzed by NAD+-dependent alcohol (ADH), aldehyde (AldDH), and formate (FDH) dehydrogenase, with regeneration of NAD+ via redox mediation to dia-phorase. (From [91], with permission from Elsevier.)... 

An immobilized-enzyme continuous-flow reactor incorporating a continuous direct electrochemical regeneration of NAD + has been proposed. To retain the low molecular weight cofactor NADH/NAD+ within the reaction system, special hollow fibers (Dow ultrafilter UFb/HFU-1) with a molecular weight cut-off of 200 has been used [32],... 

Other mediators which have been used in combination with diaphorase for the regeneration of NAD+ are riboflavin and Vitamin K3, which is 2,3-dimethyl-1,4-naphthoquinone. However, especially riboflavin is not stable enough for synthetic applications [40]. Better stability is exhibited by phenanthrolindiones as mediators. In combination with diaphorase, Ohshiro [41] showed the indirect electrochemical oxidation of cyclohexanol to cyclohexanone using the NAD+ dependent HLADH with a turnover frequency of two per hour. For an effective enzymatic synthesis, this turnover frequency, however, would be too small. In our own studies, we were able to accelerate the NAD(P)+ regeneration considerably by lowering the electron density within the... 

In a very special system, the photoelectrochemical regeneration of NAD(P)+ has been performed and applied to the oxidation of the model system cyclohexanol using the enzymes HLADH and TBADH. In this case, tris(2,2 -bipyridyl)ruthenium(II) is photochemically excited by visible light [43]. The excited Ru(II) complex acts as electron donor for AT,AT -dimethyl-4,4 -bipyridinium sulfate (MV2+) forming tris(2,2 -bipyridyl)ruthenium(III) and the MV-cation radical. The Ru(III) complex oxidizes NAD(P)H effectively thus... 

Fig. 11. Application of a double mediator system for the photoelectrochemical regeneration of NAD(P)+ in enzymatic oxidations with dehydrogenases...

For enzymatic reductions with NAD(P)H-dependent enzymes, the electrochemical regeneration of NAD(P)H always has to be performed by indirect electrochemical methods. Direct electrochemical reduction, which requires high overpotentials, in all cases leads to varying amounts of enzymatically inactive NAD-dimers generated due to the one-electron transfer reaction. One rather complex attempt to circumvent this problem is the combination of the NAD+ reduction by electrogenerated and regenerated potassium amalgam with the electrochemical reoxidation of the enzymatically inactive species, mainly NAD dimers, back to NAD+ [51]. If one-electron... 

Figure 6-1. The steps of glycolysis. Feedback inhibition of glucose phosphorylation by hexokinase, inhibition of pyruvate kinase, and the main regulatory, rate-limiting step catalyzed by phosphofructoki-nase (PFK-I) are indicated, pyruvate formation and substrate-level phosphorylation are the main outcomes of these reactions. Regeneration of NAD occurs by reduction of pyruvate to lactate during anaerobic glycolysis.

Figure 10.20 Enzymatic ketone reduction with regeneration of NAD(P)H.

This behavior could easily be mistaken for half-of-the-sites reactivity, but all four sites appear to be independent.55,57 Coenzymes also bind independently to each site. In contrast, the enzyme from Bacillus stearothermophilus is allosteri-cally regulated.44,45 A tetrameric form is stabilized by the binding of two effector molecules of fructose 1,6-bisphosphate and binds pyruvate 50 times more tightly than the dimeric form does. The turnover numbers of tetramer and dimer are the same, and so they form a V-system (Chapter 8). The build up of the glycolytic intermediate fructose 1,6-bisphosphate under anaerobic conditions thus stimulates the regeneration of NAD+. 

The electron transfer between NADH and the anode may be accelerated by the use of a mediator. Synthetic applications have been described for the oxidation of primary and secondary alcohols to aldehydes and ketones catalyzed by yeast alcohol dehydrogenase (YADH) and the alcohol dehydrogenase from Thermoanaerobium brockii (TBADH) with indirect electrochemical regeneration of NAD+ and NADP+, respectively, using the tris(3,4,7,8-tetramethyl-l,10-phenanthroline) iron(II/III) complex as redox catalyst [59],... 

Regeneration of NAD+ by reduction of pyruvate to lactate. Because NAD+ is a necessary participant in the oxidation of glyceraldehyde-3-phosphate to glycerate-l,3-bisphosphate, glycolysis is possible only if there is a way by which NADH can be reoxidized. 

B. R. Riebel, P. R. Gibbs, W. B. Wellborn, and A. S. Bommarius, Cofador regeneration of NAD+ from NADH novel water-forming NADH oxidases,... 

The most convenient and useful enzymatic methods for the regeneration of NAD(P)H are formate/formate dehydrogenase for NADH [210, 211], iso-propanol/TBADH for NADPH [57], isopropanol/ADH (Pseudomonas sp.) for NADH [61, 212] and glucose/glucose dehydrogenase (Bacillus sp.) for NADH and NADPH [213],... 

In fact, the a-ketoglutarate/glutamate dehydrogenase is a generally applicable method for the regeneration of NAD and NADP in laboratory scale productions. Both components involved are inexpensive and stable. Quite recently, a method for the oxidation of the reduced nicotinamide coenzymes based on bacterial NAD(P)H oxidase has been described [225], This enzyme oxidizes NADH as well as NADPH with low Km values. The product of this reaction is peroxide, which tends to deactivate enzymes, but it can be destroyed simultaneously by addition of catalase. The irreversible peroxide/catalase reaction favours the ADH catalyzed oxidation reaction, and complete conversions of this reaction type are described. 

Oxidative regeneration of NAD(P)+ cofactors can be accomplished by two general routes, see Fig. 37. NAD(P)H might act as an electron donor for an excited photosensitizer, thereby regenerating NAD(P)+ by a reductive ET quenching mechanism (Fig. 37 a). Alternatively, the oxidized photoproduct formed in an... 

Fig. 38. Photosensitized regeneration of NAD(P)+ cofactors using methylene blue, MB+, or N-methyl phenazonium methylsulfonate, MPMS+, as photosensitizers...

Answer Anaerobiosis requires the regeneration of NAD+ from NADH in order to allow glycolysis to continue. 

This metabolic scheme, which is called lactate fermentation, is shown in Fig. 11-7. The coreactant cycle between the two dehydrogenase enzymes, glyceraldehyde-3-phosphate dehydrogenase (Step 6) and lactate dehydrogenase, ensures that there is regeneration of NAD+ in this particular oxidation state so that glycolysis, lactate fermentation, and the production of ATP can continue. 

---