NAD-linked dehydrogenases

Figure 9.18 The glycerol phosphate shuttle. In the cytosol, NADH is oxidised in a reaction in which dihydroxyacetone phosphate is reduced to glycerol 3-phosphate, catalysed by glycerol-3-phosphate dehydrogenase (NAD linked) ...

Enzymic syntheses are considered next. Xylitol is a substrate for sheep-liver L-iditol dehydrogenase, a NAD-linked enzyme. 1-Deoxy-D-xylitol, prepared by Raney nickel reduction of D-xylose diethyl dithioacetal in a 27% overall yield from D-xylose, was also reported31 to be a substrate, although with a higher Km and lower Vmax. The product was assumed to be l-deoxy-D-f/ireo-pentulose because of the appearance of a yellowish fluorescent spot when a chromatogram was sprayed with acidic 3,5-aminobenzoic acid, resembling that formed from 1 -deoxyfructose. There was no more-rigorous characterization. 

Generally, NAD-linked dehydrogenases catalyze ox-idoreduction reactions in the oxidative pathways of metabolism, particularly in glycolysis, in the citric acid cycle, and in the respiratory chain of mitochondria. NADP-linked dehydrogenases are found characteristically in reductive syntheses, as in the extramitochon-drial pathway of fatty acid synthesis and steroid synthesis—and also in the pentose phosphate pathway. 

Nevertheless, the nature and function of this cofactor will have to be clarified by further investigations. It is possible that this problem is involved in the way by which the energy for reduction is provided, whether NADH2 or NADPH2 is oxidized by the action of MHbR (cf. G5, R12, S10, Sll, S17). This may be implicated in the results showing the enhancement of MHb reduction by aliphatic and aromatic aldehydes effected by a NAD-linked aldehyde dehydrogenase (M9, M10). 

The reactivity of xylitol in the polyol dehydrogenase reactions has been extensively studied for its relations to pentosuria. Xylitol was found to be oxidized in two ways (a) to L-xylulose by a highly specific NADP-requiring dehydrogenase and (b) to D-xylulose by a NAD-linked enzyme with lesser substrate specificity ... 

Nicotinamide Nucleotides.—A number of dehydrogenases have been purified by affinity chromatography, using NAD+ linked to insoluble supports either through the 6-11)12 or the 8-positions13 of the adenine nucleus. The reverse process, the use of immobilized dehydrogenases to purify NAD+, has also been described recently.14... 

Oxidation of Malate to Oxaloacetate In the last reaction of the citric acid cycle, NAD-linked L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate ... 

NAD-linked dehydrogenases remove two hydrogen atoms from their substrates. One of these is transferred as a hydride ion ( II ) to NAD+ the other is released as H+ in the medium (see Fig. 13-15). NADH and NADPH are water-soluble electron carriers that associate reversibly with dehydrogenases. NADH carries electrons from catabolic reactions to their point of entry into the respiratory chain, the NADH dehydrogenase complex described below. NADPH generally supplies electrons to anabolic reactions. Cells maintain separate pools of NADPH and NADH, with different redox potentials. This is accomplished by holding the ratios of [reduced form]/[oxidized form] relatively high for NADPH and relatively low for NADH. Neither NADH nor NADPH can cross the inner mitochondrial membrane, but the electrons they carry can be shuttled across indirectly, as we shall see. 

In mitochondria, hydride ions removed from substrates by NAD-linked dehydrogenases donate electrons to the respiratory (electron-transfer) chain, which transfers the electrons to molecular 02, reducing it to H20. 

Shuttle systems convey reducing equivalents from cytosolic NADH to mitochondrial NADH. Reducing equivalents from all NAD-linked dehydrogenations are transferred to mitochondrial NADH dehydrogenase (Complex I). 

This hypothesis presumes that early free-living prokaryotes had the enzymatic machinery for oxidative phosphorylation and predicts that their modern prokaryotic descendants must have respiratory chains closely similar to those of modern eukaryotes. They do. Aerobic bacteria carry out NAD-linked electron transfer from substrates to 02, coupled to the phosphorylation of cytosolic ADP. The dehydrogenases are located in the bacterial cytosol and the respiratory chain in the plasma membrane. The electron carriers are similar to some mitochondrial electron carriers (Fig. 19-33). They translocate protons outward across the plasma membrane as electrons are transferred to 02. Bacteria such as Escherichia coli have F0Fi complexes in their plasma membranes the F portion protrudes into the cytosol and catalyzes ATP synthesis from ADP and P, as protons flow back into the cell through the proton channel of F0. 

Tissues of the mammalian central nervous system contain a pyridoxal phosphate-dependent glutamate decarboxylase that catalyzes conversion of Glu to y-aminobutyrate (GABA), an inhibitory synaptic transmitter. GABA is degraded by trans-imination with a-oxoglutarate as the acceptor to yield succinic semialdehyde, which then is oxidized to succinate by an NAD-linked dehydrogenase. 

Ordered mechanisms often occur in the reactions of the NAD+-linked dehydrogenases, with the coenzyme binding first. The molecular explanation for this is that the binding of the dinucleotide causes a conformational change that increases the affinity of the enzyme for the other substrate (see Chapter 16). 

CT Hou, RN Patel, AI Laskin, N Barnabe. NAD-linked formate dehydrogenase from methanol-grown Pichia pastoris NRRL-Y-7556. Arch Biochem Biophys 216 296-305, 1982. 

The major mechanistic difference between the pro-5 and the pro-/ specific enzymes in this area where thermodynamic constraints are weak or non-existent seems to be that the pro-/ specific enzymes contain a zinc ion at the active site whereas the pro-5 specific enzymes do not (Schneider-Bernlohr et al., 1986). In the mechanism of an NAD+-linked alcohol dehydrogenase shown in Scheme 6, in the reduction direction the substrate carbonyl group was shown as polarised by partial proton donation from a Bronsted acid BH + this polarisation can equally well be achieved by coordination to an active site zinc, which acts as a Lewis acid. One thus has two mechanistic classes of enzyme, but even this difference affects the stereochemistry only in a very limited region close to the break-point. 

This means that this dehydrogenase can form binary complexes with steroid substrates, binary complexes with pyridine nucleotide, and ternary complexes with both substrates. This behavior contrasts with that usually observed for NAD-linked dehydrogenases in which the ketone or aldehyde substrate can bind only to the NADH-enzyme binary complex but not to free enzyme (29). 

The oxidation of L-glycerol 3-phosphate to dihydroxyacetone phosphate is catalyzed by two different enzymes. One is the cytoplasmic NAD-linked a-glycerophosphate dehydrogenase, and the other is the mitochondrial enzyme, which appears to contain flavin and iron. The latter enzyme was first studied by Green in 1936 (223). It was shown to be associated with respiratory particles, and widely distributed in animal tissues. The highest concentration of the enzyme was found in the brain. Lardy and co-workers (234) studied the enzyme in deoxycholate-solubilized particles obtained from skeletal muscle, confirmed the finding... 

The oxidation of choline to betaine is catalyzed by two enzymes. First, choline is oxidized to betaine aldehyde by an enzyme which is found in mitochondria in membrane-bound form. This enzyme is believed to be a flavoprotein containing nonheme iron. Betaine aldehyde is then oxidized to betaine by a soluble enzyme, which is NAD-linked. Betaine aldehyde dehydrogenase appears to be present both in mitochondria and the soluble fraction of liver 243, 246). 

Li, H.L., Moreno-Sanchez, R., Rottenherg, H. (1995). Alcohol inhibits the activation of NAD-linked dehydrogenases hy calcium in hrain and heart mitochondria. Biochim. Biophys. Acta 1236 306-16. 

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