Dinucleotide with nicotinamide, proton

DHFR catalyzes the reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) using nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor (Fig. 17.1). Specifically, the pro-R hydride of NADPH is transferred stereospecifi-cally to the C6 of the pterin nucleus with concurrent protonation at the N5 position [1]. Structural studies of DHFR bound with substrates or substrate analogs have revealed the location and orientation of H2F, NADPH and the mechanistically important side chains [2]. Proper alignment of H2F and NADPH is crucial in enhancing the rate of the chemical step (hydride transfer). Ab initio, mixed quantum mechanical/molecular mechanical (QM/MM), and molecular dynamics computational studies have modeled the hydride transfer process and have deduced optimal geometries for the reaction [3-6]. The optimal C-C distance between the C4 of NADPH and C6 of H2F was calculated to be 2.7A [5, 6], which is significantly smaller than the initial distance of 3.34 A inferred from X-ray crystallography [2]. One proposed chemical mechanism involves a keto-enol tautomerization (Fig. 

Figure 18.2 Summary of respiratory energy flows. Foods ate converted into the reduced form of nicotinamide adenine dinucleotide (NADH), a strong reductant, which is the most reducing of the respiratory electron carriers (donors). Respiration can he based on a variety of terminal oxidants, such as O2, nitrate, or fumarate. Of those, O2 is the strongest, so that aerobic respiration extracts the largest amount of free energy from a given amount of food. In aerobic respiration, NADH is not oxidized directly by O2 rather, the reaction proceeds through intermediate electron carriers, such as the quinone/quinol couple and cytochrome c. The most efficient respiratory pathway is based on oxidation of ferrocytochrome c (Fe ) with O2 catalyzed by cytochrome c oxidase (CcO). Of the 550 mV difference between the standard potentials of c)Tochrome c and O2, CcO converts 450 mV into proton-motive force (see the text for further details).

P. Mitchell (Nobel Prize for Chemistry, 1978) explained these facts by his chemiosmotic theory. This theory is based on the ordering of successive oxidation processes into reaction sequences called loops. Each loop consists of two basic processes, one of which is oriented in the direction away from the matrix surface of the internal membrane into the intracristal space and connected with the transfer of electrons together with protons. The second process is oriented in the opposite direction and is connected with the transfer of electrons alone. Figure 6.27 depicts the first Mitchell loop, whose first step involves reduction of NAD+ (the oxidized form of nicotinamide adenosine dinucleotide) by the carbonaceous substrate, SH2. In this process, two electrons and two protons are transferred from the matrix space. The protons are accumulated in the intracristal space, while electrons are transferred in the opposite direction by the reduction of the oxidized form of the Fe-S protein. This reduces a further component of the electron transport chain on the matrix side of the membrane and the process is repeated. The final process is the reduction of molecular oxygen with the reduced form of cytochrome oxidase. It would appear that this reaction sequence includes not only loops but also a proton pump, i.e. an enzymatic system that can employ the energy of the redox step in the electron transfer chain for translocation of protons from the matrix space into the intracristal space. 

Working at 3.0T with proton decoupling, Sevastianova et al. partially resolved the resonance of nicotinamide adenine dinucleotide phosphate (NADPH) from that of a-NTP. ° NADPH was higher in subjects with nonalcoholic steatohepatitis and cirrhosis than in healthy controls and correlated with disease severity. Solga et al. investigated the reliability of both and P MRS at 1.5 T in cohorts of obese diabetic subjects and healthy con-trols. ° Whereas hepatic fat could be reliably measured with relative ease, hepatic ATP proved difficult with obese subjects due to reduced P SNR. 

K. Sevastianova, A. Hakkarainen, A. Kotronen, A. Comer, P. Arkkila, J. Arola, J. Westerbacka, R. Bergholm, J. Lundbom, N. Lundbom and H. Yki-Jarvinen, Nonalcoholic fatty Uver disease detection of elevated nicotinamide adenine dinucleotide phosphate with in vivo 3.0-T P MR spectroscopy with proton decoupling. Radiology, 2010, 256,466-473. 

Alcohol dehydrogenases (ADH EC 1.1.1.1), for which several X-ray structures are available ", catalyze the biological oxidation of primary and secondary alcohols via the formal transfer of a hydride anion to the oxidized form of nicotinamide adenine dinucleotide (NAD ), coupled with the release of a proton. Liver alcohol dehydrogenase (LADH) consists of two similar subunits, each of which contains two zinc sites, but only one site within each subunit is catalytically active. The catalytic zinc is coordinated in a distorted tetrahedral manner to a histidine residue, two cysteine residues and a water molecule. The remaining zinc is coordinated tetrahedrally to four cysteine residues and plays only a structural role . 

The basicity of pyridinyl radicals is of interest since some pyridinyl radicals react with one another in an acid catalyzed reaction (sect. 4.4). The absorption spectra of radicals generated from the pyridinium ion by pulse radiolysis in aqueous solution at different pH values allow the evaluation of the basicity of the radicals 97,98) I jjg Qf protonated form of l-methyl-3-carbamidopyridinyI radical, (CONHj) ), is 1.43 the protonated radical has alsorption maxima at 3(X) nm and 440 nm, at somewhat longer wavelengths than those for the unprotonated radical at 280 nm and 420 nm. The nicotinamide adenine dinucleotide radical (NAD ) has a pK, of 0 or less, with a shift in absorption maximum due to protonation being observed only in 1.5 M HCIO4. 

With the exception of reduced -nicotinamide adenine dinucleotide (NADH), substrates interact at the Mo centre and two electrons are transferred from the substrate to Mo(VI), reducing the metal to Mo(IV). The substrate residue reacts with an oxo ligand of Mo and a proton also reduces a terminal sulphide ligand of Mo. Hydrolysis of the Mo-substrate complex releases oxidized product, while the Mo(IV) is reoxidized by intramolecular transfer to other redox centres. The catalytic mechanism as described by Bray is depicted below [23], Aldehyde oxidase and xanthine oxidase can each take up to six... 

Figure 2. Cycles of life and death. A brief overview showing how paraquat (and similarly diquat) interacts with two of the most fundamental processes of life photosynthesis in the chloroplast and respiration in the mitochondrion. Abbreviations ADP, adenosine diphosphate ATP, adenosine triphosphate e, electron proton NAD and NADH, oxidised and reduced forms of nicotinamide adenine dinucleotide NADP and NADPH, oxidised and reduced forms of nicotinamide adenine dinucleotide phosphate Paraquat, paraquat radical Pi, inorganic phosphate superoxide radical.

Reoxidation of the cosubstrate at an appropriate electrode surface will lead to the generation of a current that is proportional to the concentration of the substrate, hence the coenzyme can be used as a kind of mediator. The formal potential of the NADH/NAD couple is - 560 mV vs. SCE (KCl-saturated calomel electrode) at pH 7, but for the oxidation of reduced nicotinamide adenine dinucleotide (NADH) at unmodified platinum electrodes potentials >750 mV vs. SCE have to be applied [142] and on carbon electrodes potentials of 550-700 mV vs. SCE [143]. Under these conditions the oxidation proceeds via radical intermediates facilitating dimerization of the coenzyme and forming side-products. In the anodic oxidation of NADH the initial step is an irreversible heterogeneous electron transfer. The resulting cation radical NADH + looses a proton in a first-order reaction to form the neutral radical NAD, which may participate in a second electron transfer (ECE mechanism) or may react with NADH (disproportionation) to yield NAD [144]. The irreversibility of the first electron transfer seems to be the reason for the high overpotential required in comparison with the enzymatically determined oxidation potential. 

Figure 3.8. Structures of vitamins or vitamin-derived molecules that function in oxidation-reduction reactions. The oxidation of these redox groups in the inner mitochondricil membrane contributes to the electron transport chain that carries electrons from the oxidation of glucose to oxygen and in the process pumps protons from one side to the other of the inner mitochondrial membrane (see Chapter 8 for details). The proton gradient thus formed is used to phosphorylate ADP to form 32 of the 36 ATPs resulting from the oxidation of one glucose molecule to six CO2 and six H2O molecules. A Vitamin B3, also called niacin or nicotinic acid, becomes converted to the amide (nicotinamide) and dressed up with a ribose sugar. Then, in a manner like that of riboflavin in B becomes phosphorylated to form nicotinamide mononucleotide (NMN) or further reacted with the addition of adenosine monophosphate (AMP) to form nicotinamide adenine dinucleotide (NAD). B Vitamin B2, also known as riboflavin, is shown converted to the forms involved in redox reactions such as those of the electron transport chain. (From Biochemistry, Second Edition, D. Voet and J. Voet, Copyright 1995, John Wiley Sons, New York. Reprinted with permission of John Wiley Sons, Inc.)...

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