Pyruvic acid, reduction with NADH

In contrast, amino acid dehydrogenases comprise a well-known class of enzymes with industrial apphcations. An illustrative example is the Evonik (formerly Degussa) process for the synthesis of (S)-tert-leucine by reductive amination of trimethyl pyruvic acid (Scheme 6.12) [27]. The NADH cofactor is regenerated by coupling the reductive amination with FDH-catalyzed reduction of formate, which is added as the ammonium salt. 

Diacetyl, and its reduction products, acetoin and 2,3-butanediol, are also derived from acetaldehyde (Fig 8D.7), providing additional NADH oxidation steps. Diacetyl, which is formed by the decarboxylation of a-acetolactate, is regulated by valine and threonine availability (Dufour 1989). When assimilable nitrogen is low, valine synthesis is activated. This leads to the formation of a-acetolactate, which can be then transformed into diacetyl via spontaneous oxidative decarboxylation. Because valine uptake is suppressed by threonine, sufficient nitrogen availability represses the formation of diacetyl. Moreover, the final concentration of diacetyl is determined by its possible stepwise reduction to acetoin and 2,3-butanediol, both steps being dependent on NADH availability. Branched-chain aldehydes are formed via the Ehrlich pathway (Fig 8D.7) from precursors formed by combination of acetaldehyde with pyruvic acid and a-ketobutyrate (Fig 8D.7). 

This reaction is completely enantioselective. For example, reduction of pyruvic acid with NADH catalyzed by lactate dehydrogenase affords a single enantiomer of lactic acid with the S configuration. NADH reduces a variety of different carbonyl compounds in biological systems. The configuration of the product (/ or S) depends on the enzyme used to catalyze the process. 

Oxidoreductases are concerned with oxidation and reduction processes. Dehydrogenases form one type of these and will catalyse the removal of H atoms from a substrate and transfer them to an acceptor. Important hydrogen acceptors are nicotinamide adenine dinucleotide, NAD+, and the closely related nicotinamide adenine dinncleotide phosphate, NADP (12.23). Lactic dehydrogenase catalyses the transfer of hydrogen from lactic acid which gives pyruvic acid and converts NAD+ into the rednced form NADH (12.25). 

Glycolysis The initial pathway in the catabolism of carbohydrates, by which a molecule of glucose is broken down to two molecules of pyruvate, with a net production of ATP molecules and the reduction of two NAD molecules to NADH. Under aerobic conditions, these NADH molecules are reoxidized by the electron transport chain under anaerobic conditions, a different electron acceptor is used. An anaerobic metabolic pathway used to break down glucose into pyruvic acid while producing some ATP. 

The product of this metabolic sequence, pyruvate, is a metabolite of caitral importance. Its fate depends upon the conditions within a cell and upon the type of cell. When oxygen is plentiful pyruvate is usually converted to acetyl-coenzyme A, but under anaerobic conditions it may be reduced by NADH + H+ to the alcohol lactic acid (Fig. 10-3, step h). This reduction exactly balances the previous oxidation step, that is, the oxidation of glycer-aldehyde 3-phosphate to 3-phospho-glycerate (steps a and b). With a balanced sequence of an oxidation reaction, followed by a reduction reaction, glucose can be converted to lactate in the absence of oxygen, a fermentation process. The lactic acid fermentation occurs not only in certain bacteria but also in our own muscles under conditions of extremely vigorous exercise. It also occurs continuously in some tissues, e.g., the transparent lens and cornea of the eye. 

The details of the process and the oxidation-reduction balance can be pictured as in Eq. 17-25. Pyruvate is cleaved by the pyruvate formate-lyase reaction (Eq. 15-37) to acetyl-CoA and formic acid. Half of the acetyl-CoA is cleaved to acetate via acetyl-P with generation of ATP, while the other half is reduced in two steps to ethanol using the two molecules of NADH produced in the initial oxidation of triose phosphate (Eq. 17-25). The overall energy yield is three molecules of ATP per glucose. The "efficiency" is thus (3 x 34.5)... 

Figure 7-1. Pathways of fuel metabolism and oxidative phosphorylation. Pyruvate may be reduced to lactate in the cytoplasm or may be transported into the mitochondria for anabolic reactions, such as gluconeogenesis, or for oxidation to acetyl-CoA by the pyruvate dehydrogenase complex (PDC). Long-chain fatty acids are transported into mitochondria, where they undergo [ -oxidation to ketone bodies (liver) or to acetyl-CoA (liver and other tissues). Reducing equivalents (NADH, FADII2) are generated by reactions catalyzed by the PDC and the tricarboxylic acid (TCA) cycle and donate electrons (e ) that enter the respiratory chain at NADH ubiquinone oxidoreductase (Complex 0 or at succinate ubiquinone oxidoreductase (Complex ID- Cytochrome c oxidase (Complex IV) catalyzes the reduction of molecular oxygen to water, and ATP synthase (Complex V) generates ATP fromADP Reprinted with permission from Stacpoole et al. (1997).

L-Lactate dehydrogenase (l-LDH, EC 1.1.1.27) catalyzes the reduction of pyruvate to (S)-lactate with a simultaneous oxidation of NADH. l-LDH is found in all higher organisms. There are two kinds of l-LDHs enzymes from one group are activated by fructose 1,6-diphosphate while the other group stays independent [71]. l-LDH is highly selective for pyruvate, short-chain 2-keto acids and phenylpyruvic acid [80]. All bacterial NAD+-dependent LDHs form lactate from pyruvate in vivo, and there is no evidence at all that they catalyze the other direction as well. The equilibrium constant lies far on the direction of lactate formation, and thus the reaction catalyzed by bacterial LDHs can be considered almost irreversible. LDHs from some lacto-bacilli like Lactobacillus fermentum or L. cellobiosus show no or just poor reaction with lactate [71], whereas mammalian LDHs can be considered as reversible [71]. Well characterized l-LDHs are summarized in Table 2. 

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