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PubMed1984

Kinetic properties of NAD malic enzyme from cauliflower.

The kinetic characteristics of NAD malic enzyme purified to homogeneity from cauliflower florets have been examined. Free NAD+ is the active form of this coenzyme. Double-reciprocal plots of data obtained by varying NAD+ and malate2- at a saturating concentration of Mg2+ or by varying Mg2+ and NAD+ at a saturating level of malate2-are of intersecting type. This indicates that NAD malic enzyme obeys a sequential mechanism. Analysis of these sets of data suggests that each of these substrate pairs binds randomly to the enzyme. However, each substrate binds tighter when others are already present on the enzyme. NAD malic enzyme cannot decarboxylate malate2- in the absence of either Mg2+ or NAD+. Arrhenius plots of the NAD-linked reaction are concave downward, indicating the existence of two rate-determining steps with activation energies of 26.5 and 14.2 kcal/mol, respectively. In addition to Mg2+, the enzyme can also use Mn2+ and Co2+. Using Co2+ in place of Mg2+ does not change Vmax or Km, malate2- but the Km for metal and NAD+ are greatly decreased. At pH 7.0 and above, Mn2+ isotherms and malate2- curves with Mn2+ are nonlinear and appear to be composed of two separate saturation curves. NAD malic enzyme is completely and irreversibly inactivated by N-ethylmaleimide. The enzyme is also irreversibly inactivated approximately 50% by KCNO.

Ethylmaleimide↗
PubMed2005

Identification of the mitochondrial NAD+ transporter in Saccharomyces cerevisiae.

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and cofactors across the inner mitochondrial membrane. In Saccharomyces cerevisiae, NAD+ is synthesized outside the mitochondria and must be imported across the permeability barrier of the inner mitochondrial membrane. However, no protein responsible for this transport activity has ever been isolated or identified. In this report, the identification and functional characterization of the mitochondrial NAD+ carrier protein (Ndt1p) is described. The NDT1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported NAD+ and, to a lesser extent, (d)AMP and (d)GMP but virtually not alpha-NAD+, NADH, NADP+, or NADPH. Transport was saturable with an apparent Km of 0.38 mM for NAD+. The Ndt1p-GFP was found to be targeted to mitochondria. Consistently with Ndt1p localization and its function as a NAD+ transporter, cells lacking NDT1 had reduced levels of NAD+ and NADH in their mitochondria and reduced activity of mitochondrial NAD+-requiring enzymes. Similar results were also found in the mitochondria of cells lacking NDT2 that encodes a protein (Ndt2p) displaying 70% homology with Ndt1p. The delta ndt1 delta ndt2 double mutant exhibited lower mitochondrial NAD+ and NADH levels than the single deletants and a more pronounced delay in growth on nonfermentable carbon sources. The main role of Ndt1p and Ndt2p is to import NAD+ into mitochondria by unidirectional transport or by exchange with intramitochondrially generated (d)AMP and (d)GMP.

Adenosine Monophosphate↗
PubMed2005

First archaeal inorganic polyphosphate/ATP-dependent NAD kinase, from hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization.

The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90 degrees C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95 degrees C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.

Adenosine Triphosphate↗
PubMed2004

NAD(P)H fluorescence imaging of mitochondrial metabolism in contracting Xenopus skeletal muscle fibers: effect of oxygen availability.

The blue autofluorescence (351 nm excitation, 450 nm emission) of single skeletal muscle fibers from Xenopus was characterized to be originating from mitochondrial NAD(P)H on the basis of morphological and functional correlations. This fluorescence signal was used to estimate the oxygen availability to isolated single Xenopus muscle fibers during work level transitions by confocal microscopy. Fibers were stimulated to generate two contractile periods that were only different in the PO2 of the solution perfusing the single fibers (PO2 of 30 or 0-2 Torr; pH = 7.2). During contractions, mean cellular NAD(P)H increased significantly from rest in the low PO2 condition with the core (inner 10%) increasing to a greater extent than the periphery (outer 10%). After the cessation of work, NAD(P)H decreased in a manner consistent with oxygen tensions sufficient to oxidize the surplus NAD(P)H. In contrast, NAD(P)H decreased significantly with work in 30 Torr PO2. However, the rate of NAD(P)H oxidation was slower and significantly increased with the cessation of work in the core of the fiber compared with the peripheral region, consistent with a remaining limitation in oxygen availability. These results suggest that the blue autofluorescence signal in Xenopus skeletal muscle fibers is from mitochondrial NAD(P)H and that the rate of NAD(P)H oxidation within the cell is influenced by extracellular PO2 even at high extracellular PO2 during the contraction cycle. These results also demonstrate that although oxygen availability influences the rate of NAD(P)H oxidation, it does not prevent NAD(P)H from being oxidized through the process of oxidative phosphorylation at the onset of contractions.

Animals↗
PubMed1976

Demonstration and possible function of NADH:NAD+ transhydrogenase from ascaris muscle mitochondria.

Mitochondria from the muscle of Ascaris lumbricoides var. suis function anaerobically. NADH is generated in the intermembrane space as a consequence of the "malic" enzyme reaction. It has been suggested that this reducing equivalent in the form of hydride ion, would be translocated across the inner membrane in order to mediate ATP generation via the fumarate reductase reaction. In accord with this suggestion, intact Ascaris mitochondria showed appreciable NADH oxidase activity. Sonication resulted in an approximately 2-fold increase in NADH oxidase activity, whereas "malic" enzyme, fumarase, and NADH:NAD+ transhydrogenase activities increased approximately 7- to 14-fold, respectively. Phosphorylation capabilities and permeability toward pyridine nucleotides also indicated the intactness of the mitochondria. Ascaris mitochondria incubated anaerobically in the presence of fumarate, and [14C]NADH catalyzed a rapid reduction of the fumarate to succinate with the concomitant formation of equivalent quantities of extramitochondrial NAD+. However, very little isotope was recovered from the washed mitochondria, indicating the possibility of hydride ion translocation in the absence of nucleotide translocation. NADH:NAD+ transhydrogenase has been isolated from the muscle mitochondria of the intestinal nematode, Ascaris lumbricoides var. suis. The enzyme seems to have been solubilized from the mitochondrial membrane fraction by treatment with sodium deoxycholate followed by dialysis and subsequent adsorption by and elution from alumina C gamma. No NADPH:NAD+ transhydrogenase activity was detectable, making the Ascaris system unique over others reported. Activity was protected by L-cysteine, reduced glutathione and dithioerythritol, but strongly inhibited by low concentrations of p-chloromercuribenzoate or silver nitrate. The thionicotinamide derivative of NAD+ (thioNAD+) was employed to accept hydride ions from NADH in order to assay spectrophotometrically at 398 nm. Apparent Km values for thioNAD+ and NADH were 1 X 10(-4) M and 8 X 10(-6) M, respectively. That the physiological nucleotide, could act as hydride ion acceptor from NADH was indicated by the findings that NAD+ competitively inhibited the reduction of thioNAD+ when assayed at 398 nm. The additional finding of a noncompetitive inhibition between NAD+ and NADH suggested at least two binding sites on the enzyme, one for NADH and another common site for NAD+ and thioNAD+. More conclusive evidence indicating the participation of NAD+ as acceptor was obtained by incubation of the enzyme with NADH and [14C]NAD+ and demonstrating a rapid formation of [14C]NADH. These findings, in conjunction with those discussed above, suggest a physiological function of this enzyme in hydride ion translocation.

Aerobiosis↗
PubMed1997

Detection and identification of NAD-catabolizing activities in rat tissue homogenates.

NAD may be degraded in several ways. A large number of investigations have shown that at least those catabolic routes which involve the formation of ADP-ribose are related to regulatory processes. In this study a rapid assay was utilized that permits identification of NAD-degrading enzymes directly in sodium dodecylsulfate polyacrylamide gels. Enzymatic activities were recovered by washing the gels in the presence of mild detergents such as lauryl dimethylamine N-oxide or Triton X-100. Subsequent incubation of the gels in the presence of the fluorescent analog 1,N6 etheno-NAD visualized NAD-degrading enzymes. Following excision of the fluorescent bands from the gels, the actual activity of the proteins was established by incubating the gel slices with 14C-labeled NAD and subsequent product analysis by thin layer chromatography (TLC). Homogenates from rat renal cortex and spleen were analyzed by this procedure. While in the spleen homogenate only a single band could be 'activity-stained', in the kidney three bands were detected. Kidney proteins with apparent molecular masses of about 210,000 and 105,000 Da were identified as phosphodiesterase and NAD pyrophosphatase (alkaline phosphodiesterase I), respectively. The third protein exhibited an apparent molecular mass of 41,000. The spleen protein (apparent molecular mass 45,000 Da) cleaved NAD to nicotinamide and ADP-ribose identifying it as NAD glycohydrolase. The procedure is suitable to screen for NAD-converting activities in crude extracts. It is specific for proteins which function as monomers or homo-oligomers.

Adenosine Diphosphate Ribose↗
PubMed1990

Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+.

The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity.

Macromolecular Substances↗
PubMed2001

A self-restricted CD38-connexin 43 cross-talk affects NAD+ and cyclic ADP-ribose metabolism and regulates intracellular calcium in 3T3 fibroblasts.

Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD(+) transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD(+) to the universal calcium mobilizer cyclic ADP-ribose (cADPR). Here we demonstrate that functional uncoupling between CD38 and Cx43 in CD38-transfected 3T3 murine fibroblasts is paralleled by decreased [Ca(2+)](i) levels as a result of reduced intracellular conversion of NAD(+) to cADPR. A sharp inverse correlation emerged between [Ca(2+)](i) levels and NAD(+) transport (measured as influx into cells and as efflux therefrom), both in the CD38(+) cells (high [Ca(2+)](i), low transport) and in the CD38(-) fibroblasts (low [Ca(2+)](i), high transport). These differences were correlated with distinctive extents of Cx43 phosphorylation in the two cell populations, a lower phosphorylation with high NAD(+) transport (CD38(-) cells) and vice versa (CD38(+) cells). Conversion of NAD(+)-permeable Cx43 to the phosphorylated, NAD(+)-impermeable form occurs via Ca(2+)-stimulated protein kinase C (PKC). Thus, a self-regulatory loop emerged in CD38(+) fibroblasts whereby high [Ca(2+)](i) restricts further Ca(2+) mobilization by cADPR via PKC-mediated disruption of the Cx43-CD38 cross-talk. This mechanism may avoid: (i) leakage of NAD(+) from cells; (ii) depletion of intracellular NAD(+) by CD38; (iii) overproduction of intracellular cADPR resulting in potentially cytotoxic [Ca(2+)](i).

3T3 Cells↗
PubMed1984

[ADP-ribosylation of histones of the chicken liver nucleus at different rates of glycohydrolase hydrolysis of NAD].

The rate of incorporation of nicotinamide-[adenosine-U-14C]adenine dinucleotide [( Ado-U-14C]NAD) into histones and the poly(ADPR) polymerase activity of chromatin suggest that the NAD-dependent ADP-ribosylation of histones depends on the rate of NAD hydrolysis by glycohydrolase in chicken liver nuclei. With a rise in the NAD-glycohydrolase activity after treatment of nuclei with Triton X-100 the synthesis of poly(ADP-ribose) via the poly(ADPR)polymerase reaction is augmented, as a result of which the rate of [Ado-U-14C]NAD incorporation into total histones is increased. On the contrary, the decrease of NAD-glycohydrolase hydrolysis after treatment of nuclei with SDS lowers the poly(ADPR)polymerase activity and [Ado-U-14C]NAD incorporation into histones. Under these conditions, i. e. different rates of glycohydrolase hydrolysis of NAD in the nuclei, some redistribution of [Ado U-14C]NAD incorporation into individual histones occurs.

Animals↗
PubMed1998

Oral L-glutamine therapy for sickle cell anemia: I. Subjective clinical improvement and favorable change in red cell NAD redox potential.

Previously, we demonstrated that there is an increased utilization of glutamine by intact sickle red blood cells (RBC) in conjunction with nicotinamide adenine dinucleotide (NAD) metabolism in vitro. In this report, we describe the in vivo effect of L-glutamine supplementation on total NAD, nicotinamide adenine dinucleotide reduced (NADH), and NAD redox potential of sickle RBC. Seven adult sickle cell anemia patients participated in this study. The exclusion criteria were pregnancy, previous or current use of hydroxyurea, and transfusion within 3 months of initiation of the study. After proper consent, L-glutamine was started at a dose of 30 g/day administered orally. Fasting blood samples were drawn at baseline and after 4 weeks of therapy by routine phlebotomy for evaluation of RBC total NAD and NADH levels. We found significant changes in both the NADH level and NAD redox potential (ratio of NADH to NAD+ + NADH). NAD redox potential increased from 47.2 +/- 3.7% to 62.1 +/- 11.8% (P < 0.01). The NADH level increased from 47.5 +/- 6.3 to 72.1 +/- 15.1 nmol/ml RBC (P < 0.01). The total NAD level demonstrated an upward trend (from 101.2 +/- 16 to 116.4 +/- 14.7 nmol/ml RBC) but this was not statistically significant. Our data show that oral L-glutamine can significantly increase the NAD redox potential and NADH level in sickle RBC. These changes may decrease oxidative susceptibility of sickle RBC and result in clinical benefit.

Administration, Oral↗
PubMedDate not supplied

Conformations of nicotinamide adenine dinucleotide (NAD(+)) in various environments.

Enzymes bind NAD(+) in extended conformations and yet NAD(+) exists in aqueous solution as a compact, folded molecule. Thus, NAD(+) conformation is environment dependent. In an attempt to investigate the effects of environmental changes on the conformation of NAD(+), a series of molecular dynamics simulations in different solvents was performed. The solvents investigated (water, DMSO, methanol and chloroform) represented changes in relative permittivity and hydrophobic character. The simulations predicted folded conformations of NAD(+) to be more stable in water, DMSO and methanol. In contrast, extended conformations of NAD(+) were observed to be more stable in chloroform. Furthermore, the extended conformations observed in chloroform were similar to conformations of NAD(+) bound to enzymes. In particular, a large separation between the aromatic rings and a strong interaction between the pyrophosphate and nicotinamide groups were observed. The implications of these observations for the recognition of NAD(+) by enzymes is discussed. It is argued that a hydrophobic environment is important for stabilizing unfolded conformations of NAD(+).

Dimethyl Sulfoxide↗
PubMedDate not supplied

Liver parenchyma heterogeneity in the response to extracellular NAD+.

The perfused rat liver responds intensely to NAD+ infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption are some of the effects that were observed. The aim of the present work was to investigate the distribution of the response to extracellular NAD+ along the hepatic acinus. The bivascularly perfused rat liver was used. Various combinations of perfusion directions (antegrade and retrograde) and infusion routes (portal vein, hepatic vein and hepatic artery) were used in order to supply NAD+ to different regions of the liver parenchyma, also taking advantage of the fact that its extracellular transformation generates steep concentration gradients. Oxygen uptake was stimulated by NAD+ in retrograde perfusion (irrespective of the infusion route) and transiently inhibited in antegrade perfusion. This indicates that the signal causing oxygen uptake inhibition is generated in the periportal area. The signal responsible for oxygen uptake stimulation is homogenously distributed. Stimulation of glucose release was more intense when NAD+ was infused into the portal vein or into the hepatic artery, indicating that stimulation of glycogenolysis predominates in the periportal area. The increases in perfusion pressure were more pronounced when the periportal area was supplied with NAD+ suggesting that the vasoconstrictive elements responding to NAD+ predominate in this region. The response to extracellular NAD+ is thus unequally distributed in the liver. As a paracrine agent, NAD+ is likely to be released locally. It can be concluded that its effects will be different depending on the area where it is released.

Animals↗
PubMed2002

CD38 expression and NAD+-induced intracellular Ca+ mobilization in isolated retinal Müller cells.

Müller cells of the vertebrate retina are prominent radial glia that provide essential support to sustain homeostasis of the tissue, including redistribution of external potassium, uptake and metabolism of neurotransmitters, and secretion of factors that stabilize the retina. Meeting this diversity of functional supports requires that Müller cells express numerous receptors, transporters, enzymes, and tissue factors. In this study, we provide evidence that adds to the dimensions of Müller cell function by demonstrating a unique relationship between external NAD(+) and the mobilization of internal calcium, expressed in the form of calcium waves. The cellular mechanism that supports internal mobilization of calcium appears to depend on a complex multifunctional ectoenzyme, CD38, which converts NAD(+) into the intracellular Ca(2+)-mobilizing second-messenger cyclic ADP-ribose (cADPR) and could function as a detector for extracellular NAD(+), thus providing a novel signal detection system for evaluating the extracellular environment. Our results are consistent with a model of intracellular Ca(2+) mobilization in which membrane-bound CD38 binds extracellular NAD(+) and triggers intracellular Ca(2+) waves either by direct conversion of NAD(+) to cADPR or by activating intracellular cADPR synthesis. Our preliminary results indicate that the Ca(2+) waves induced by external NAD(+) propagate through an internal pathway that depends on the activation of ryanodine receptors, which appear to be distributed throughout the Müller cell cytosol. Because NAD(+) is likely to be enhanced when cells are stress or damaged, CD38 could enable Müller cells to detect NAD(+) under these circumstances and respond appropriately. Alternatively, NAD(+) could also represent a novel extracellular, paracrine function that mediates signaling between glial cells and/or other cellular elements of the retina.

ADP-ribosyl Cyclase↗
PubMed1994

The NAD kinase: a phosphoryltransferase displaying an oxido-reductase activity--an electrophoretic study.

The search for a valuable technique for rapid detection, after electrophoresis, of the activity of various NAD kinase isoforms possibly present in different plant materials, has revealed interesting peculiarities of this enzyme (EC 2.7.1.23; also called ATP:NAD+ 2'-phosphotransferase). At first and in the unique but obligatory presence of NAD, the NAD kinase acts almost instantaneously as an oxido-reductase (probably coupled with the transformation of NAD to NADH). In the additional presence of ATP, the transformation of NAD+ to NADP+ reinforced such an oxido-reductase activity. Final assays testing for the specificity of the phosphoryl donor revealed that not only ATP but also GTP, G6P, and even NADP could be the substrate; the efficiencies of these phosphoryl donors varied with the different isoforms of NAD kinase, evidenced in the different seeds tested, and compared with NAD kinase from heterotropically grown Euglena cells, and NAD kinase purified from chicken liver (from Sigma Chemical Co.).

Adenosine Triphosphate↗
PubMed1999

Peroxisome-proliferator regulates key enzymes of the tryptophan-NAD+ pathway.

Structually diverse peroxisome-proliferators (PPs) were investigated regarding their effects on NAD+ level and two key enzyme activities in the tryptophan (Trp)-NAD+ pathway in the liver of rats (Sprague-Dawley male) fed PP-containing diets freely for 2 weeks. All PPs, except for thyroxine, significantly increased hepatic NAD+ level in concert with hepatic hypertrophy. Activity of quinolinate phosphoribosyltransferase (QAPRTase), one of the key enzymes in the Trp-NAD+ pathway, was increased by the PPs which caused significant increase in the hepatic NAD+. On the other hand, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSDase), another key enzyme in the Trp-NAD+ pathway, was drastically inhibited by all PPs except for linolenic acid, which was only slightly inhibitory. Most PPs investigated activated peroxisomal marker enzymes such as palmitoyl-CoA oxidase, catalase, and PPAR-alpha(peroxisome-proliferator activated receptor-alpha)-dependent enzymes, such as malic enzyme and l-3-glycerophosphate dehydrogenase. NAD+ was also increased in the rat hepatocytes cultured in the medium supplemented with PPs. These data suggested that regulation of the key enzymes in the Trp-NAD+ pathway was associated with PPAR-alpha directly or indirectly, and as a consequence the hepatic NAD+ was increased by PPs.

Animals↗
PubMed1987

The cell-cycle dependent and the DNA-damaging agent-induced changes of cellular NAD content and their significance.

NAD is the substrate of a novel chromatin-associated enzyme-ADP-ribosyl transferase (ADPRT). In this study, the cell-cycle dependent change in cellular NAD content was observed in a line of human amnion FL cells. It was found that the cellular NAD content of FL cells was highest in G1 and lowest in S/G2-G2. 3AB, a potent ADPRT inhibitor, can inhibit the cell cycle dependent change in cellular NAD content and also inhibit DNA synthesis in the S phase and extend the S phase. The results indicate that ADP-ribosylation may be involved in DNA replication and cell cycle progression. It was also found that the DNA-damaging agents, MNNG, MMS and 4NQO could lower cellular NAD content in a dose-dependent way. This DNA-damage-induced NAD lowering could be partially or completely prevented by the ADPRT inhibitors, 3AB or nicotinamide, which were shown to exert no influence on either the cellular NAD content of normal quiescent FL cells or the metabolic blocking agent, 2,4-DNP-induced cellular NAD lowering. The possibility of establishing a simple and specific method to detect DNA-damaging agents by measuring cellular NAD content in the presence or absence of ADPRT inhibitor is explored.

4-Nitroquinoline-1-oxide↗
PubMed1988

Properties of a coenzyme, pyrroloquinoline quinone: generation of an active oxygen species during a reduction-oxidation cycle in the presence of NAD(P)H and O2.

The oxidation of NAD(P)H by pyrroloquinoline quinone (PQQ) was non-enzymatically carried out at physiological pH in the presence of O2. The PQQ-NAD(P)H system requires about 1 mol of O2 for the oxidation of 1 mol of NAD(P)H. The oxidation of NAD(P)H occurred at a pseudo-first-order rate with respect to NAD(P)H and was of zero order with respect to PQQ concentration in in the presence of O2: k0[PQQ] [NAD(P)H] = k1 [NAD(P)H], where k0[PQQ] = k1, in which [PQQ] represents the initial concentration of PQQ. k0 values for NADH and NADPH were 3.4.10(2) M-1.min-1 and 2.0.10(2) M-1.min-1, respectively, at 25 degrees C and at 258 microM O2 (initial concentration). The system produced O-2, probably by the interaction of PQQ.H and/or NAD(P).with O2, during the oxidation of NAD(P)H. PQQH2 and PQQ.H were easily oxidized to PQQ in the presence of O2, yielding H2O2.

Coenzymes↗
PubMed2005

Key NAD+-binding residues in human 15-hydroxyprostaglandin dehydrogenase.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins, and is believed to be the key enzyme responsible for the biological inactivation of these biologically potent eicosanoids. The enzyme utilizes NAD(+) specifically as a coenzyme. Potential amino acid residues involved in binding NAD(+) and facilitating enzyme catalysis have been partially identified. In this report, we propose that three more residues in 15-PGDH, Ile-17, Asn-91, and Val-186, are also involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine their roles in binding NAD(+). Several mutants (I17A, I17V, I17L, I17E, I17K, N91A, N91D, N91K, V186A, V186I, V186D, and V186K) were prepared, expressed as glutathione S-transferase (GST) fusion enzymes in Escherichia coli, and purified by GSH-agarose affinity chromatography. Mutants I17E, I17K, N91L, N91K, and V186D were found to be inactive. Mutants N91A, N91D, V186A, and V186K exhibited comparable activities to the wild type enzyme. However, mutants I17A, I17V, I17L, and V186I had higher activity than the wild type. Especially, the activities of I17L and V186I were increased nearly 4- and 5-fold, respectively. The k(cat)/K(m) ratios of all active mutants for PGE(2) were similar to that of the wild type enzyme. However, the k(cat)/K(m) ratios of mutants I17A and N91A for NAD(+) were decreased 5- and 10-fold, respectively, whereas the k(cat)/K(m) ratios of mutants I17V, N91D, V186I, and V186K for NAD(+) were comparable to that of the wild type enzyme. The k(cat)/K(m) ratios of mutants I17L and V186A for NAD(+) were increased over nearly 2-fold. These results suggest that Ile-17, Asn-91, and Val-186 are involved in the interaction with NAD(+) and contribute to the full catalytic activity of 15-PGDH.

Amino Acid Sequence↗