Stereospecificity of C4 nicotinamide hydrogen transfer of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The stereospecificity of the reaction catalysed by the spinach chloroplast enzyme NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) with respect to the C4 nicotinamide hydrogen transfer was investigated. NADPH deuterated at the C4 HA position was synthesized using aldehyde dehydrogenase. 1H-NMR spectroscopy was used to examine the NADP+ product of the GPDH reaction for the presence or absence of the C4 deuterium atom. Chloroplast NADP-dependent glyceraldehyde-3-phosphate dehydrogenase retains the deuterium at the C4 HA position (removing the hydrogen atom), and is therefore a B (pro-S) specific dehydrogenase.     

Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis.

A-Side (4-R)-(4-²H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d₆ to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1-d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final $\text{A}{}_{260}\text{A}{}_{340}$ ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.     

ATP-driven electron transfer in enzymatic radical reactions

A novel method to generate organic radicals in enzymatic reactions is described, which is similar to electron transfer in nitrogenase. Component A of 2-hydroxyglutaryl-CoA dehydratase contains a [4Fe-4S] cluster located at the interface between its two identical subunits. The cluster is reduced by one electron derived from ferredoxin or flavodoxin. Hydrolysis of two ATP bound to component A, one to each subunit, enhances the reductive power of the electron and transfers it to component D, the actual dehydratase, where a low potential [4Fe-4S](2+) cluster is probably reduced. Further transfer to the substrate (R)-2-hydroxyglutaryl-CoA probably generates a substrate-derived ketyl radical anion, which expels the adjacent hydroxyl group. The resulting enoxy radical is deprotonated to a product-related ketyl radical anion. Finally the electron is removed by the next incoming substrate leading to the product glutaconyl-CoA and starting a new turnover. A similar, but stoichiometric rather than catalytic electron transfer has been established for the related benzoyl-CoA reductase.     

The localization of enzymatic activities involved in uridine nucleotides reactions in human erythrocytes

Enzymatic activities involved in the transformations of uridine nucleotides by intact erythrocytes and their subfractions have been studied and the following enzymatic activities have been identified: UTPase, UDPase, UMPase and uridylate kinase. UTPase activity was present exclusively in the stromal fraction while UMPase and uridylate kinase activities were specific in the soluble fraction. UDPase was present in both the stromal and soluble fractions. This compartmentation in erythrocytes differs from that reported for the enzymes involved in adenine nucleotides transformations. Due to its sensitivity to Zn²⁺, uridylate kinase could be differentiated from adenylate kinase.     

Linkage of nanosecond protein motion with enzymatic methyl transfer by nicotinamide N-methyltransferase

Nicotinamide N-methyltransferase (NNMT), a key cytoplasmic protein in the human body, is accountable to catalyze the nicotinamide (NCA) N¹-methylation through S-adenosyl-L-methionine (SAM) as a methyl donor, which has been linked to many diseases. Although extensive studies have concerned about the biological aspect, the detailed mechanism study of the enzyme function, especially in the part of protein dynamics is lacking. Here, wild-type nicotinamide N-methyltransferase together with the mutation at position 20 with Y20F, Y20G, and free tryptophan were carried out to explore the connection between protein dynamics and catalysis using time-resolved fluorescence lifetimes. The results show that wild-type nicotinamide N-methyltransferase prefers to adapt a less flexible protein conformation to achieve enzyme catalysis.     

Ketopantoic acid and ketopantoyl lactone reductases. Stereospecificity of transfer of hydrogen from reduced nicotinamide adenine dinucleotide phosphate.

The stereospecificity of hydrogen transfer from NADPH to the appropriate carbonyl substrate catalyzed by ketopantoic acid and ketopantoyl acid and ketopantoyl lactone reductases of yeast (Saccharomyces cerevisiae) and Escherichia coli has been determined. Yeast and E. coli ketopantoic acid reductases are B-specific enzymes which transfer hydrogen from [4B-3H]-NADPH to ketopantoic acid to form [3H]pantoic acid. In contrast to the usual observations on the stereospecificity of functionally similar dehydrogenases from different species, yeast and E. coli ketopantoyl lactone reductases exhibit opposite stereospecificities. Both of two forms of yeast ketopantoyl lactone reductases are A-specific enzymes which form [3H]pantoyl lactone from ketopantoyl lactone and [4A-3H]NADPH, whereas, two forms of E. coli ketopantoyl lactone reductases are B-specific enzymes.     

Isotope effects in the study of enzymatic phosphoryl transfer reactions

CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, binds to the carboxyl-terminus of beta-neurexins on the intracellular side of the presynaptic membrane. The guanylate kinase-like (GUK) domains of MAGUKs lack kinase activities, but might be important for mediating specific protein-protein interaction. By a yeast two-hybrid approach, we identified an interaction between the GUK domain of CASK and the C2B domain of rabphilin3a, a presynaptic protein involved in synaptic vesicle exocytosis. The interaction was confirmed by in vitro GST pull-down and co-immunoprecipitation assays. It was proposed that presynaptic vesicles might be guided to the vicinity of points of exocytosis defined by beta-neurexins via the interaction between rabphilin3a-CASK-beta-neurexins.     

Oxidative changes in brain pyridine nucleotides and neuroprotection using nicotinamide

Pyridine nucleotides are critical during oxidative stress due to their roles in reductive reactions and energetics. The aim of the present study was to examine pyridine nucleotide changes in six brain regions of mice after an intracerebroventricular injection of the oxidative stress inducing agent, t-butyl hydroperoxide (t-BuOOH). A secondary aim was to investigate the correlation between NAD+ levels and DNA fragmentation. Here, we demonstrate that t-BuOOH induced a rapid oxidation of NADPH and a slow depletion of NAD+ in most brain regions. A slight increase in NADH also occurred in five brain regions. NAD+ depletion was associated with increased DNA fragmentation. This suggests the initiation of a death cascade involving poly(ADP-ribose) polymerase (PARP), NAD+, ATP depletion and consequent cell death in brain tissue. PARP activity was accelerated in some brain regions after 20 min of oxidative stress. To counteract oxidative stress induced toxicity, NAD+ levels were increased in the brain using an intraperitoneal injection of nicotinamide. A surplus of brain NAD+ prevented DNA fragmentation in some brain regions. Nicotinamide administration also resulted in higher brain NADH, NADP+ and NADPH levels in some regions. Their synthesis was further upregulated during oxidative stress. Nicotinamide as a precursor for NAD+ may provide a useful therapeutic strategy in the treatment of neurodegeneration.     

Adenylate and nicotinamide nucleotides in developing soybean seeds during seed-fill

Profiles of adenylate and nicotinamide nucleotides in soybean seeds were determined during seed-fill. The ATP content per seed increased during the early seed-filling stages to a level of 10 to 12 micrograms per seed. Seed ATP decreased after 40 days of development and reached its lowest level of less than 1 microgram at maturity. The ATP:ADP ratios were relatively constant at all seed development stages. Sharp increases in AMP levels during the late seed-fill stages were paralleled with a disappearance of ATP and ADP pools resulting in a reduced seed energy charge. Energy charge varied from the highest value of 0.78 at mid-seed-fill to less than 0.10 at maturity.     

Energy transfer and molecular switching II. Muscle contraction and enzymatic reactions

In Part I (Barrett, 1981), the concept of chemical parametric excitation was reviewed and applied to the process of nerve action potential excitation and regeneration. In the present paper, the chemical reactions involved in muscle contraction and the enzymatic reaction are examined and shown to be examples of chemical parametric excitation.It is demonstrated that in a model biochemical scheme for an enzymatic reaction, the enzyme is activated from a state, X, to a state, $\text{X}{}^1$, and in this activated state pumps the reaction parametrically. The concept of enzyme is identified with an excited state or state of disequilibrium permitting a release of energy during the dissipation, $\text{X}{}^1\text{→X}$, in the enzymatic reaction, which is powered by the release of energy in the return to the unexcited state X. The demonstration of parametric excitation relations for chemical systems indicates an explanation for the directionality of energy flow and designates an energy pumping role for an enzyme.In muscle contraction, the role of $\text{X}{}^1$ is played by actomyosin and Ca²⁺, and the enzymatic reaction is the hydrolysis of ATP. The release of energy caused by this hydrolysis reaction brings about the conformational changes underlying muscle contraction.     

Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer

Kinetic parameters and primary deuterium kinetic isotope effects for NADH and five pyridine nucleotide substrates have been determined at pH 8.1 for human erythrocyte glutathione reductase. DV/KNADH and DV are equal to 1.4 and are pH independent below pH 8.1, but DV decreases to 1.0 at high pH as a group exhibiting a pK of 8.6 is deprotonated. This result suggests that as His-467' is deprotonated, the rate of the isotopically insensitive oxidative half-reaction is specifically decreased and becomes rate-limiting. For all substrates, equivalent V and V/K primary deuterium kinetic isotope effects are observed at pH values below 8.1. The primary deuterium kinetic isotope effect on V, but not V/K, is sensitive to solvent isotopic composition. The primary tritium kinetic isotope effects agree well with the corresponding value calculated from the primary deuterium kinetic isotope effects by using the Swain-Schaad relationship. This suggests that the primary deuterium kinetic isotope effects observed in these steady-state experiments are the intrinsic primary deuterium kinetic isotope effects for hydride transfer. The magnitude of the primary deuterium kinetic isotope effect is dependent on the redox potential of the pyridine nucleotide substrate used, varying from approximately 1.4 for NADH and -320 mV reductants to 2.7 for thioNADH to 4.2-4.8 for 3-acetylpyridine adenine dinucleotide (3APADH). The alpha-secondary tritium kinetic isotope effects also increase as the redox potential of the pyridine nucleotide substrate becomes more positive. Together, these data indicate that the transition state for hydride transfer is very early for NADH and becomes later for thioNADH and 3APADH, as predicted by Hammond's postulate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diurnal changes in adenylates and nicotinamide nucleotides in sugar beet leaves

Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers at 25°C, with a photon flux density of 500 mol m^-2s^-1. Measurements were made of net CO₂ exchange, leaf adenylates (ATP, ADP and AMP), and leaf nicotinamide nucleotides (NAD⁺, NADP⁺, NADH, NADPH), over the diurnal period (16h light/8 h dark) and during photosynthetic induction. All the measurements were carried out on recently expanded leaves from 5-week-old plants. When the lights were switched on in the growth chamber, the rate of photosynthetic CO₂ uptake, and the levels of leaf ATP and NADPH increased to a maximum in 30 min and remained there throughout the light period. The increase in ATP over the first few minutes of illumination was associated with the phosphorylation of ADP to ATP and the increase in NADPH with the reduction of NADP⁺; subsequently, the increase in ATP was associated with an increase in total adenylates while the increase in NADPH was associated with an accumulation of NADP⁺ and NADPH due to the light-driven phosphorylation of NAD⁺ to NADP⁺. On return to darkness, ATP and NADPH values decreased much more slowly, requiring 2 to 4 hours to reach minimum values. From these results we suggest that (i) the total adenylate and NADPH and NADP⁺ (but not NAD⁺ and NADH) pools increase following exposure to light; (ii) the increase in pool size is not accompanied by any large change in the energy or redox states of the system; and (iii) the measured ratios of ATP/ADP and NADPH/NADP⁺ for intact leaves are low and constant during steady-state illumination.Abbreviations AEC adenylate energy charge - DHAP dihydroxyacetone phosphate - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide - PES phenazine ethosulfate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - PFD photon flux density - Ru5P ribulose-5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Determination of the hydride transfer stereospecificity of nicotinamide adenine dinucleotide linked oxidoreductases by proton magnetic resonance.

A facile proton magnetic resonance technique is described for the determination of the coenzyme stereospecificity during hydride transfer reactions catalyzed by pyridine nucleotide dependent oxidoreductases. The reliability of this technique was demonstrated by examining the coenzyme stereospecificity of lactate, malate, and 3-phosphoglycerate dehydrogenases, which are known to be A-stereospecific enzymes, as well as triosephosphate and octopine dehydrogenases, which are known to be B-stereospecific enzymes. Furthermore, by applying this technique, it was shown that the previously unstudied enzymes D-beta-hydroxybutyrate and 4-aminobutanal dehydrogenases are B- and A-stereospecific enzymes, respectively. In addition, the nicotinamide adenine dinucleotide linked reaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be B stereospecific, like the reaction of the nicotinamide adenine dinucleotide phosphate linked yeast enzyme.     

Pyridine nucleotides in normal and nicotinamide depleted adrenal tumor cell cultures

Mouse adrenal tumor cell line Y-129 was grown in cell culture in medium without nicotinamide. Inhibition of growth occurred after the second subculture in the vitamin-deficient medium. Pyridine nucleotides were measured in control and nicotinamide-starved cultures. DPN⁺ decreased to less than 10% of the normal level after 7 days of vitamin starvation. TPNH dropped to 25% of its normal level in the same period. Plating efficiency decreased from 20% in controls to 10% in 7-day nicotinamide-starved cultures. Steroid production was reduced by approximately 50% in the starved cultures. Sensitivity of the cultures to the lethal effects of the nicotinamide antagonist, 3-acetylpyridine, was inversely proportional to the pyridine nucleotide content of the cells. Reconstitution of nicotinamide in depleted cultures caused a rapid renewal of the pyridine nucleotide levels and complete protection against the effects of 3-acetylpyridine. Recovery and subsequent growth of the cultures did not restore the decreased capacity to produce steroids. Nicotinic acid was not utilized by the cells for the synthesis of pyridine nucleotides in depleted cultures and azaserine did not inhibit the utilization of nicotinamide indicating that DPN⁺ was synthesized directly via nicotinamide ribonucleotide.     

Stereospecificity of hydride transfer in NAD+-catalyzed 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics

The key enzyme in the biosynthesis of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which converts ubiquitous d-glucose 6-phosphate (G-6-P) into the specific carbocycle, 2-deoxy-scyllo-inosose with an aid of NAD(+)-NADH recycling. The NAD(+)-dependent first step of the DOIS reaction was examined in detail by the use of 6-phosphonate and 6-homophosphonate analogs of G-6-P. Both analogs showed competitive inhibition against the DOIS reaction with K(i) values of 1.3 and 2.8 mM, respectively, due to their inability for the subsequent phosphate elimination. Based on the direct spectrophotometric observation of NADH formed by the hydride transfer from 6-phosphonate to NAD(+), the stereospecificity of the hydride transfer in the DOIS reaction was analyzed with 6-[4-(2)H]phosphonate and was found to be pro-R specific.     

A new technique for determining the hydride transfer stereospecificity of nicotinamide adenine dinucleotide-linked oxidoreductases: Electron impact and field desorption mass spectrometry

The stereospecificity of nicotinamide adenine dinucleotide-linked oxidoreductases can be determined directly without additional purification and with high sensitivity by low-resolution electron impact mass spectrometry and low-resolution field desorption mass spectrometry. The ease and reliability of this new technique is demonstrated by studying the hydride transfer stereospecificity of lactate dehydrogenase from pig heart (known to be A-specific) and glutamate dehydrogenase from beef liver (known to be B-specific). In addition the stereospecificity of glutamate dehydrogenases from duckweed (Lemna minor) was found to be B-specific. The sample quantity of the unpurified products needed is about 1 μg. The time required for one mass spectrometric determination was about 30 min.