Biological Oxidation-Reduction Reactions:- NADH and NADPH Act with Dehydrogenases as Soluble Electron Carriers
Nicotinamide adenine dinucleotide (NAD in its oxidized form) and its close analog nicotinamide adenine dinucleotide phosphate (NADP ) are composed of two nucleotides joined through their phosphate groups by a phosphoanhydride bond (Fig. 13–15a). Because the nicotinamide ring resembles pyridine, these compounds are sometimes called pyridine nucleotides. The vita min niacin is the source of the nicotinamide moiety in nicotinamide nucleotides. Both coenzymes undergo reversible reduction of the nicotinamide ring (Fig. 13–15). As a substrate molecule undergoes oxidation (dehydrogenation), giving up two hydrogen atoms, the oxidized form of the nucleotide (NAD or NADP+) accepts a hydride ion (:H+, the equivalent of a proton and two electrons) and is trans formed into the reduced form (NADH+ or NADPH+). The second proton removed from the substrate is released to the aqueous solvent. The half-reaction for each type of nucleotide is therefore
Reduction of NAD+ or NADP+ converts the benzenoid ring of the nicotinamide moiety (with a fixed positive charge on the ring nitrogen) to the quinonoid form (with no charge on the nitrogen). Note that the reduced nucleotides absorb light at 340 nm; the oxidized forms do not (Fig. 13–15b). The plus sign in the abbreviations NAD+ and NADP+ does not indicate the net charge on these molecules (they are both negatively charged); rather, it indicates that the nicotinamide ring is in its oxidized form, with a positive charge on the nitrogen atom. In the abbreviations NADH and NADPH, the “H” denotes the added hydride ion. To refer to these nucleotides without specifying their oxidation state, we use NAD and NADP.
The total concentration of NAD+ tissues is about 10-5 M; that of NADP+ NADH in most NADPH is about 10-6 M. In many cells and tissues, the ratio of NAD+ (oxidized) to NADH (reduced) is high, favoring hydride transfer from a substrate to NAD to form NADH. By contrast, NADPH (reduced) is generally present in greater amounts than its oxidized form, NADP+, favoring hydride transfer from NADPH to a substrate. This reflects the specialized metabolic roles of the two coenzymes: NAD generally functions in oxidations— usually as part of a catabolic reaction; and NADPH is the usual coenzyme in reductions—nearly always as part of an anabolic reaction. A few enzymes can use ei ther coenzyme, but most show a strong preference for one over the other. The processes in which these two cofactors function are also segregated in specific organelles of eukaryotic cells: oxidations of fuels such as pyruvate, fatty acids, and -keto acids derived from
FIGURE 13–15 NAD and NADP. (a)Nicotinamide adenine dinucleotide, NAD+, and its phosphorylated analog NADP+ undergo re duction to NADH and NADPH, accepting a hydride ion (two electrons and one proton) from an oxidizable substrate. The hydride ion is added to either the front (the A side) or the back (the B side) of the planar nicotinamide ring (see Table 13–8). (b)The UV absorption spectra of NAD and NADH. Reduction of the nicotinamide ring produces a new, broad absorption band with a maximum at 340 nm. The pro duction of NADH during an enzyme-catalyzed reaction can be conveniently followed by observing the appearance of the absorbance at 340 nm (the molar extinction coefficient ∑ 340 6,200 M-1cm-1).
amino acids occur in the mitochondrial matrix, whereas reductive biosynthesis processes such as fatty acid syn thesis take place in the cytosol. This functional and spatial specialization allows a cell to maintain two distinct pools of electron carriers, with two distinct functions. More than 200 enzymes are known to catalyze re actions in which NAD+ (or NADP+) accepts a hydride ion from a reduced substrate, or NADPH (or NADH) do nates a hydride ion to an oxidized substrate. The general reactions are
where AH2 is the reduced substrate and A the oxidized substrate. The general name for an enzyme of this type is oxidoreductase; they are also commonly called de hydrogenases. For example, alcohol dehydrogenase catalyzes the first step in the catabolism of ethanol, in which ethanol is oxidized to acetaldehyde:
Notice that one of the carbon atoms in ethanol has lost a hydrogen; the compound has been oxidized from an alcohol to an aldehyde (refer again to Fig. 13–13 for the oxidation states of carbon).
When NAD+ or NADP+ is reduced, the hydride ion could in principle be transferred to either side of the nicotinamide ring: the front (A side) or the back (B side), as represented in Figure 13–15a. Studies with iso topically labeled substrates have shown that a given en zyme catalyzes either an A-type or a B-type transfer, but not both. For example, yeast alcohol dehydrogenase and lactate dehydrogenase of vertebrate heart transfer a hydride ion to (or remove a hydride ion from) the A side of the nicotinamide ring; they are classed as type A de hydrogenases to distinguish them from another group of enzymes that transfer a hydride ion to (or remove a hydride ion from) the B side of the nicotinamide ring (Table 13–8). The specificity for one side or another can be very striking; lactate dehydrogenase, for example, prefers the A side over the B side by a factor of 5x107! Most dehydrogenases that use NAD or NADP bind the cofactor in a conserved protein domain called the Rossmann fold (named for Michael Rossmann, who deduced the structure of lactate dehydrogenase and first described this structural motif). The Rossmann fold typically consists of a six-stranded parallel sheet and four associated helices (Fig. 13–16).
The association between a dehydrogenase and NAD+ or NADP is relatively loose; the coenzyme readily diffuses from one enzyme to another, acting as a water-soluble carrier of electrons from one metabolite to another. For example, in the production of alcohol during fermentation of glucose by yeast cells, a hydride ion is removed from glyceraldehyde 3-phosphate by one enzyme (glyceraldehyde 3-phosphate dehydrogenase, a type B en zyme) and transferred to NAD+. The NADH produced then leaves the enzyme surface and diffuses to another enzyme (alcohol dehydrogenase, a type A enzyme), which transfers a hydride ion to acetaldehyde, producing ethanol:
FIGURE 13–16 The nucleotide binding domain of the enzyme lactate dehydrogenase. (a) The Rossmann fold is a structural motif found in the NAD-binding site of many dehydrogenases. It consists of a six-stranded parallel β sheet and four α helices; inspection reveals the arrangement to be a pair of structurally similar β-α-β-α-β motifs. (b) The dinucleotide NAD binds in an extended conformation through hydrogen bonds and salt bridges (derived from PDB ID 3LDH).
(1) Glyceraldehyde 3-phosphate NAD+ → 3-phosphoglycerate+NADH+H+
(2) Acetaldehyde +NADH +H+ →ethanol +NAD+
Notice that in the overall reaction there is no net pro duction or consumption of NAD or NADH; the coenzymes function catalytically and are recycled repeatedly without a net change in the concentration of NAD++ NADH.
