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PubMed1999

Amelioration by glucose-6-phosphate and NADP of potato glycoalkaloid inhibition in cell, enzyme and liposome assays.

Lysis of human erythrocytes by 20 microM chaconine was reduced by 0.5 mM glucose-6-phosphate (G6P) and NADP. Both compounds caused approximately 50% inhibition of haemolysis at 1 mM. Glucose, glucose-1-phosphate, rhamnose, galactose and galactose-6-phosphate were ineffective; NAD was effective, although not to the extent of NADP. Of the tested sugars, only G6P reduced solanine-induced haemolysis. G6P also reduced the synergistic haemolytic action of solanine and chaconine in combination. G6P and NADP at or above 5 mM antagonised chaconine-induced betanin loss from excised red beet root discs; NADP was more effective than G6P. Disruption of PC/cholesterol liposomes by chaconine and inhibition of acetylcholinesterase by chaconine or solanine, were unaffected by up to 10 mM NADP or 50 mM G6P.

Acetylcholinesterase↗
PubMed2000

Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. RESULTS: We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. CONCLUSIONS: The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

Amino Acid Sequence↗
PubMed1996

The structure of glucose-fructose oxidoreductase from Zymomonas mobilis: an osmoprotective periplasmic enzyme containing non-dissociable NADP.

BACKGROUND: The organism Zymomonas mobilis occurs naturally in sugar-rich environments. To protect the bacterium against osmotic shock, the periplasmic enzyme glucose-fructose oxidoreductase (GFOR) produces the compatible, solute sorbitol by reduction of fructose, coupled with the oxidation of glucose to gluconolactone. Hence, Z mobilis can tolerate high concentrations of sugars and this property may be useful in the development of an efficient microbial process for ethanol production. Each enzyme subunit contains tightly associated NADP which is not released during the catalytic cycle. RESULTS: The structure of GFOR was determined by X-ray crystallography at 2.7 A resolution. Each subunit of the tetrameric enzyme comprises two domains, a classical dinucleotide-binding domain, and a C-terminal domain based on a predominantly antiparallel nine-stranded beta sheet. In the tetramer, the subunits associate to form two extended 18-stranded beta sheets, which pack against each other in a face to face fashion, creating an extensive interface at the core of the tetramer. An N-terminal arm from each subunit wraps around the dinucleotide-binding domain of an adjacent subunit, covering the adenine ring of NADP. CONCLUSIONS: In GFOR, the NADP is found associated with a classical dinucleotide-binding domain in a conventional fashion. The NADP is effectively buried in the protein-subunit interior as a result of interactions with the N-terminal arm from an adjacent subunit in the tetramer, and with a short helix from the C-terminal domain of the protein. This accounts for NADP's inability to dissociate. The N-terminal arm may also contribute to stabilization of the tetramer. The enzyme has an unexpected structural similarity with the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PD). We hypothesize that both enzymes have diverged from a common ancestor. The mechanism of catalysis is still unclear, but we have identified a conserved structural motif (Glu-Lys-Pro) in the active site of GFOR and G6PD that may be important for catalysis.

Amino Acid Sequence↗
PubMed1999

Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction.

BACKGROUND: NADP-dependent malate dehydrogenase (EC 1.1.1.82) is a light-activated chloroplast enzyme that functions in the C4 pathway of photosynthesis. The light regulation is believed to be mediated in vivo by thioredoxin-catalyzed reduction and re-oxidation of cystine residues. The rates of reversible activation and inactivation of the enzyme are strongly influenced by the coenzyme substrates that seem to ultimately determine the steady-state extent of activation in vivo. RESULTS: The X-ray structure of the inactive, oxidized enzyme was determined at 2.8 A resolution. The core structure is homologous to AND-dependent malate dehydrogenases. Two surface-exposed and thioredoxin-accessible disulfide bonds are present, one in the N-terminal extension and the other in the C-terminal extension. The C-terminal peptide of the inactive, oxidized enzyme is constrained by its disulfide bond to fold into the active site over NADP+, hydrogen bonding to the catalytic His225 as well as obstructing access of the C4 acid substrate. Two loops flanking the active site, termed the Arg2 and Trp loops, that contain the C4 acid substrate binding residues are prevented from closing by the C-terminal extension. CONCLUSIONS: The structure explains the role of the C-terminal extension in inhibiting activity. The negative C terminus will interact more strongly with the positively charged nicotinamide of NADP+ than NADPH, explaining why the coenzyme-binding affinities of the enzyme differ so markedly from those of all other homologous alpha-hydroxy acid dehydrogenases. NADP+ may also slow dissociation of the C terminus upon reduction, providing a mechanism for the inhibition of activation by NADP+ but not NADPH.

Amino Acid Sequence↗
PubMed1991

Conformation of NADP+ bound to a type II dihydrofolate reductase.

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs. These plasmid-derived DHFRs are responsible for confering trimethoprim resistance to the host strain. A derivative of R388 DHFR, RBG200, has been cloned and its physical properties have been characterized. This enzyme has been shown to transfer the pro-R hydrogen of NADPH to its substrate, dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases [Brito, R. M. M., Reddick, R., Bennett, G. N., Rudolph, F. B., & Rosevear, P. R. (1990) Biochemistry 29,9825]. Two distinct binary RBG200.NADP+ complexes were detected. Addition of NADP+ to RBG200 DHFR results in formation of an initial binary complex, conformation I, which slowly interconverts to a second more stable binary complex, conformation II. The binding of NADP+ to RBG200 DHFR in the second binary complex was found to be weak, KD = 1.9 +/- 0.4 mM. Transferred NOEs were used to determine the conformation of NADP+ bound to RBG200 DHFR. The initial slope of the NOE buildup curves, measured from the intensity of the cross-peaks as a function of the mixing time in NOESY spectra, allowed interproton distances on enzyme-bound NADP+ to be estimated. The experimentally measured distances were used to define upper and lower bound distance constraints between proton pairs in distance geometry calculations. All NADP+ structures consistent with the experimental distance bounds were found to have a syn conformation about the nicotinamide-ribose (X = 94 +/- 26 degrees) and an anti conformation about the adenine-ribose (X = -92 +/- 32 degrees) glycosidic bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed2002

A covalent modification of NADP+ revealed by the atomic resolution structure of FprA, a Mycobacterium tuberculosis oxidoreductase.

FprA is a mycobacterial oxidoreductase that catalyzes the transfer of reducing equivalents from NADPH to a protein acceptor. We determined the atomic resolution structure of FprA in the oxidized (1.05 A resolution) and NADPH-reduced (1.25 A resolution) forms. The comparison of these FprA structures with that of bovine adrenodoxin reductase showed no significant overall differences. Hence, these enzymes, which belong to the structural family of the disulfide oxidoreductases, are structurally conserved in very distant organisms such as mycobacteria and mammals. Despite the conservation of the overall fold, the details of the active site of FprA show some peculiar features. In the oxidized enzyme complex, the bound NADP+ exhibits a covalent modification, which has been identified as an oxygen atom linked through a carbonylic bond to the reactive C4 atom of the nicotinamide ring. Mass spectrometry has confirmed this assignment. This NADP+ derivative is likely to form by oxidation of the NADP+ adduct resulting from nucleophilic attack by an active-site water molecule. A Glu-His pair is well positioned to activate the attacking water through a mechanism analogous to that of the catalytic triad in serine proteases. The NADP+ nicotinamide ring exhibits the unusual cis conformation, which may favor derivative formation. The physiological significance of this reaction is presently unknown. However, it could assist with drug-design studies in that the modified NADP+ could serve as a lead compound for the development of specific inhibitors.

Amino Acid Sequence↗
PubMed2006

Crystal structure of NADP(H)-dependent 1,5-anhydro-D-fructose reductase from Sinorhizobium morelense at 2.2 A resolution: construction of a NADH-accepting mutant and its application in rare sugar synthesis.

Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium morelense S-30.7.5 was crystallized in complex with the cofactor NADP(H) and its structure determined to 2.2 A resolution using selenomethionine SAD (refined R(work) and R(free) factors of 18.9 and 25.0%, respectively). As predicted from the sequence and shown by the structure, AFR can be assigned to the GFO/IDH/MocA protein family. AFR consists of two domains. The N-terminal domain displays a Rossmann fold and contains the cofactor binding site. The intact crystals contain the oxidized cofactor NADP(+), whose attachment to the cofactor binding site is similar to that of NADP(+) in glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. Due to variations in length and sequence within loop regions L3 and L5, respectively, the adenine moiety of NADP(+) adopts a different orientation in AFR caused by residue Arg38 forming hydrogen bonds with the 2'-phosphate moiety of NADP(+) and cation-pi stacking interactions with the adenine ring. Amino acid replacements in AFR (S10G, A13G, and S33D) showed that Ala13 is crucial for the discrimination between NADPH and NADH and yielded the A13G variant with dual cosubstrate specificity. The C-terminal domain contains the putative substrate binding site that was occupied by an acetate ion. As determined by analogy to GFOR and by site-directed mutagenesis of K94G, D176A, and H180A, residues Lys94, Asp176, and His180 are most likely involved in substrate binding and catalysis, as substitution of any of these residues resulted in a significant decrease in k(cat) for 1,5-AF. In this context, His180 might serve as a general acid-base catalyst by polarizing the carbonyl function of 1,5-AF to enable the transfer of the hydride from NADPH to the substrate. Here we present the first structure of an AFR enzyme catalyzing the stereoselective reduction of 1,5-AF to 1,5-anhydro-d-mannitol, the final step of a modified anhydrofructose pathway in S. morelense S-30.7.5. We also emphasize the importance of the A13G variant in biocatalysis for the synthesis of 1,5-AM and related derivatives.

Adenine↗
PubMed2004

Interaction of Ferredoxin-NADP(+) Reductase with its Substrates: Optimal Interaction for Efficient Electron Transfer.

Electron transfer (ET) reactions in systems involving proteins require an oriented interaction between electron donor and acceptor in order to accommodate their respective redox centres in optimal orientation for efficient ET. Such type of reactions are critical for the maintenance of the physiological functions of living organisms, since they are implicated in vital actions, as is, for example, in the photosynthetic ET chain that leads to NADPH reduction. In this particular case, a small redox protein ET chain is responsible for ET from Photosystem I (PS I) to NADP(+). In this system the enzyme responsible for NADP(+) reduction is ferredoxin-NADP(+) reductase (FNR), a FAD-containing NADP(+) dependent reductase. In order to produce such reduction, this enzyme receives electrons from a [2Fe-2S] plant-type ferredoxin (Fd), which is previously reduced by PS I. Moreover, in the case of some algae and cyanobacteria, an FMN-dependent protein, flavodoxin (Fld), has been shown to replace Fd in this function. The processes of interaction and ET between FNR and all of its substrates involved in the photosynthetic ET chain, namely Fd, Fld and NADP(+)/H have been extensively investigated in recent years using a large number of techniques, including the introduction of site-specific mutations in combination with kinetic and structural studies of the produced mutants. The present manuscript summarises the information so far reported for an efficient interaction between FNR and its substrates, compares such information with that revealed by other systems for which the FNR structure is a prototype and, finally, discusses the implications of the processes of association in ET between FNR and its substrates.

FNR↗
PubMed1987

Cytosolic ratios of free [NADPH]/[NADP+] and [NADH]/[NAD+] in mouse pancreatic islets, and nutrient-induced insulin secretion.

When the extracellular concentration of glucose was raised from 3 mM to 7 mM (the concentration interval in which beta-cell depolarization and the major decrease in K+ permeability occur), the cytosolic free [NADPH]/[NADP+] ratio in mouse pancreatic islets increased by 29.5%. When glucose was increased to 20 mM, a 117% increase was observed. Glucose had no effect on the cytosolic free [NADH]/[NAD+] ratio. Neither the cytosolic free [NADPH]/[NADP+] ratio nor the corresponding [NADH]/[NAD+] ratio was affected when the islets were incubated with 20 mM-fructose or with 3 mM-glucose + 20 mM-fructose, although the last-mentioned condition stimulated insulin release. The insulin secretagogue leucine (10 mM) stimulated insulin secretion, but lowered the cytosolic free [NADPH]/[NADP+] ratio; 10 mM-leucine + 10 mM-glutamine stimulated insulin release and significantly enhanced both the [NADPH]/[NADP+] ratio and the [NADH]/[NAD+] ratio. It is concluded that the cytosolic free [NADPH]/[NADP+] ratio may be involved in coupling beta-cell glucose metabolism to beta-cell depolarization and ensuing insulin secretion, but it may not be the sole or major coupling factor in nutrient-induced stimulation of insulin secretion.

Animals↗
PubMed1972

Photoreduction of NADP + sensitized by synthetic pigment systems.

Two synthetic pigment systems capable of enzymatically photoreducing NADP(+) are described. One system contains proflavine; the other, acridine. The complete system consists of ethylene diamine tetraacetic acid (EDTA), proflavine (or acridine), ferredoxin-NADP reductase (EC 1.6.99.4), and NADP(+). The two pigments initiate the photoreduction of NADP(+) in different portions of the electromagnetic spectrum. Proflavine photosensitizes in the visible portion; acridine, in the ultraviolet. Neither proflavine nor acridine is structurally related to chlorophyll. The acridine system has the attractive property that the enzyme, ferredoxin-NADP reductase, is the only component of the system that absorbs appreciably in the visible region of the spectrum.

Acridines↗
PubMed1995

A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose.

We have previously shown that alkaline treatment of NADP generates a derivative which can mobilize Ca2+ from sea urchin egg homogenates (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568). In this study, the active derivative was purified and shown by high pressure liquid chromatography to be distinct from NADP and NADPH. However, its proton NMR spectrum was virtually identical to that of NADP. The mass of its molecular ion was measured by high resolution mass spectrometry to be 743.0510, one mass unit larger than the corresponding ion of NADP. These results are consistent with the active derivative being nicotinic acid adenine dinucleotide phosphate (NAADP). Ca2+ release induced by NAADP was saturable with a half-maximal concentration of about 30 nM. The release was specific since NADP and nicotinic acid adenine dinucleotide were ineffective even at 10-40-fold higher concentrations. The NAADP-dependent Ca2+ release showed desensitization and was insensitive to heparin and a specific antagonist of cyclic ADP-ribose (cADPR), 8-amino-cADPR. The release mechanism did not require calmodulin. This is similar to the inositol trisphosphate-sensitive release but distinct from that of cADPR. That the NAADP-sensitive Ca2+ stores were different from those sensitive to inositol trisphosphate- or cADPR was further indicated by their differences in distribution on Percoll density gradients. Microinjection of NAADP into live sea urchin eggs induced transient elevation of intracellular Ca2+ and triggered the cortical reaction, indicating the NAADP-dependent mechanism is operative in intact cells.

Adenosine Diphosphate Ribose↗
PubMed2001

Probing the determinants of coenzyme specificity in ferredoxin-NADP+ reductase by site-directed mutagenesis.

On the basis of sequence and three-dimensional structure comparison between Anabaena PCC7119 ferredoxin-NADP(+) reductase (FNR) and other reductases from its structurally related family that bind either NADP(+)/H or NAD(+)/H, a set of amino acid residues that might determine the FNR coenzyme specificity can be assigned. These residues include Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235. Systematic replacement of these amino acids was done to identify which of them are the main determinants of coenzyme specificity. Our data indicate that all of the residues interacting with the 2'-phosphate of NADP(+)/H in Anabaena FNR are not involved to the same extent in determining coenzyme specificity and affinity. Thus, it is found that Ser-223 and Tyr-235 are important for determining NADP(+)/H specificity and orientation with respect to the protein, whereas Arg-224 and Arg-233 provide only secondary interactions in Anabaena FNR. The analysis of the T155G FNR form also indicates that the determinants of coenzyme specificity are not only situated in the 2'-phosphate NADP(+)/H interacting region but that other regions of the protein must be involved. These regions, although not interacting directly with the coenzyme, must produce specific structural arrangements of the backbone chain that determine coenzyme specificity. The loop formed by residues 261-268 in Anabaena FNR must be one of these regions.

Amino Acid Sequence↗
PubMed2001

NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, kinetic properties, and physiological roles.

In the yeast Saccharomyces cerevisiae, two NADP(+)-dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and alpha-ketoglutarate. The GDH2-encoded NAD(+)-dependent glutamate dehydrogenase degrades glutamate producing ammonium and alpha-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in alpha-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1- and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of alpha-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of alpha-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.

Base Sequence↗
PubMed1985

Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae.

The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.

Acetates↗
PubMed1977

On the role of ferredoxin and ferredoxin-NADP+ reductase in cyclic electron transport of spinach chloroplasts.

Antibodies prepared against purified spinach ferredoxin and ferredoxin-NADP+ reductase were used as specific inhibitors of electron-transfer reactions dependent on either ferredoxin or ferredoxin-NADP+ reductase; The possible role of both electron carriers in cyclic electron transport was checked using cytochrome b6 photoreactions as indicator. It could be demonstrated that the ferredoxin antibody inhibits cytochrome b6 photoreduction. Ferredoxin-NADP+ reductase, however, appears not to be involved in this pathway: reductase antibody in concentrations sufficient to completely inhibit electron transport to NADP+ had no effect on cytochrome b6 photoreduction. Quantitative treatment of the immunoassay data showed that osmotically shocked chloroplasts contain both bound ferredoxin and ferredoxin-NADP+ reductase in concentration approximately equal to that of cytochrome b6.

Chloroplasts↗
PubMed1979

Activities of NAD-specific and NADP-specific isocitrate dehydrogenases in rat-liver mitochondria. Studies with D-threo-alpha-methylisocitrate.

The contributions of NAD-specific and NADP-specific isocitrate dehydrogenases to isocitrate oxidation in isolated intact rat liver mitochondria were examined using DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) to specifically inhibit flux through NADP-specific isocitrate dehydrogenase. Under a range of conditions tested with respiring mitochondria, the rate of isocitrate oxidation was decreased by about 20--40% by inhibition of NADP-isocitrate dehydrogenase, and matrix NADP became more oxidized. (a) For mitochondria incubated with externally added DL-isocitrate and citrate, the rate of isocitrate oxidation obtained by extrapolation to infinite alpha-methylisocitrate concentration was approximately 70% of the uninhibited rate in both state 3 and state 4. (b) With pyruvate plus malate added as substrates of citric acid cycle oxidation and isocitrate generated intramitochondrially, a concentration of alpha-methylisocitrate (400 microM) sufficient for 99.99% inhibition of NADP-isocitrate dehydrogenase inhibited isocitrate oxidation in states 4 and 3 by 21 +/- 6% and 19 +/- 11% (mean +/- SEM), respectively. (c) With externally added isocitrate and citrate, the addition of NH4Cl increased isocitrate oxidation by 3--4-fold, decreased NADPH levels by 30--40% and 2-oxoglutarate accumulation by about 40%. The further addition of 600 microM alpha-methylisocitrate decreased the NH4Cl-stimulated isocitrate oxidation by about 40% and decreased NADPH to about 30% of the level prevailing in the absence of NH4Cl; nevertheless, the rate of isocitrate oxidation was still twice as large in the presence of NH4Cl and alpha-methylisocitrate as in their absence. Experiments were also performed with intact mitochondria incubated with respiratory inhibitors to determine additional factors which might affect the flux through the two isocitrate dehydrogenases. (a) In the coupled reduction of acetoacetate by isocitrate, where the rate of reoxidation of reduced pyridine nucleotides is limited by NAD-specific 3-hydroxybutyrate dehydrogenase, 85--100% of the rate of 3-hydroxybutyrate formation was retained in the presence of 400--900 microM alpha-methylisocitrate. (b) In a system where the rate of isocitrate oxidation is limited by the rate of NADPH reoxidation by glutathione reductase, the rate of glutathione reduction extrapolated to infinite alpha-methylisocitrate concentration was from 20--40% of the uninhibited rate. (c) In the coupled synthesis of glutamate from isocitrate and NH4Cl, where the reoxidation of NADPH and NADH can occur via glutamate dehydrogenase, the rate of glutamate production extrapolated to infinite alpha-methylisocitrate concentration was about 60% of the uninhibited rate.

Ammonium Chloride↗
PubMed1980

NADP-dependent isocitrate dehydrogenase from the mussel Mytilus edulis L. 2. Kinetic studies.

The kinetic mechanism for NADP-dependent isocitrate dehydrogenase from the digestive gland of the mussel Mytilus edulis has been investigated. Initial-rate studies and substrate-analogue and product-inhibition patterns are consistent with a rapid-equilibrium random-ordered reaction mechanism, both with respect to substrate addition and product release. Product inhibition by NADPH is non-competitive versus D-isocitrate concentrations. 2-Oxoglutarate inhibition is competitive versus D-isocitrate at all NADP concentrations. The inhibition by 2-oxoglutarate versus NADP is either non-competitive or uncompetitive. Tricarballylic acid (a substrate analogue of D-isocitric acid) gives competitive inhibition versus D-isocitrate at all NADP levels and mixed inhibition versus NADP, at sub-saturating levels of D-isocitrate.

Animals↗
PubMed1985

Preparation and characterization of NADP derivatives alkylated at 2'-phosphate and 6-amino groups.

Reaction of NADP with 3-propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2'-phosphate or 6-amino group, or both: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), and 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III). Their structures were assigned on the basis of ultraviolet, 1H-NMR and 31P-NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase. Carbodiimide-promoted reaction of derivative I with 1,2-diaminoethane gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV); derivative III gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-N6-[N-(2-aminoethyl ) carbamoylethyl]-NAD (IV). The same reaction of derivative II, on the other hand, gave a mixture of N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va) and its 3'-phosphate isomer (Vb). The mixture was converted to Va via the 2',3'-cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H-NMR spectra, and also treatment with alkaline phosphatase or 3'-nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose-6-phosphate dehydrogenase.

Alkylating Agents↗