Catalytic mechanism and interactions of NAD+ with glyceraldehyde-3-phosphate dehydrogenase: correlation of EPR data and enzymatic studies

Perdeuterated spin label (DSL) analogs of NAD+, with the spin label attached at either the C8 or N6 position of the adenine ring, have been employed in an EPR investigation of models for negative cooperativity binding to tetrameric glyceraldehyde-3-phosphate dehydrogenase and conformational changes of the DSL-NAD+-enzyme complex during the catalytic reaction. C8-DSL-NAD+ and N6-DSL-NAD+ showed 80 and 45% of the activity of the native NAD+, respectively. Therefore, these spin-labeled compounds are very efficacious for investigations of the motional dynamics and catalytic mechanism of this dehydrogenase. Perdeuterated spin labels enhanced spectral sensitivity and resolution thereby enabling the simultaneous detection of spin-labeled NAD+ in three conditions: (1) DSL-NAD+ freely tumbling in the presence of, but not bound to, glyceraldehyde-3-phosphate dehydrogenase, (2) DSL-NAD+ tightly bound to enzyme subunits remote (58 A) from other NAD+ binding sites, and (3) DSL-NAD+ bound to adjacent monomers and exhibiting electron dipolar interactions (8-9 A or 12-13 A, depending on the analog). Determinations of relative amounts of DSL-NAD+ in these three environments and measurements of the binding constants, K1-K4, permitted characterization of the mathematical model describing the negative cooperativity in the binding of four NAD+ to glyceraldehyde-3-phosphate dehydrogenase. For enzyme crystallized from rabbit muscle, EPR results were found to be consistent with the ligand-induced sequential model and inconsistent with the pre-existing asymmetry models. The electron dipolar interaction observed between spin labels bound to two adjacent glyceraldehyde-3-phosphate dehydrogenase monomers (8-9 or 12-13 A) related by the R-axis provided a sensitive probe of conformational changes of the enzyme-DSL-NAD+ complex. When glyceraldehyde-3-phosphate was covalently bound to the active site cysteine-149, an increase in electron dipolar interaction was observed. This increase was consistent with a closer approximation of spin labels produced by steric interactions between the phosphoglyceryl residue and DSL-NAD+. Coenzyme reduction (DSL-NADH) or inactivation of the dehydrogenase by carboxymethylation of the active site cysteine-149 did not produce changes in the dipolar interactions or spatial separation of the spin labels attached to the adenine moiety of the NAD+. However, coenzyme reduction or carboxymethylation did alter the stoichiometry of binding and caused the release of approximately one loosely bound DSL-NAD+ from the enzyme. These findings suggest that ionic charge interactions are important in coenzyme binding at the active site.     

Differential binding of NAD+ to acyl glyceraldehyde-3-phosphate dehydrogenase and its role in the acyl group transfer reaction.

Enzyme protein fluorescence of di-furylacryloyl-glyceraldehyde-3-phosphate dehydrogenase (di-FA-GPDH:lambda max.excitation 290 nm, lambda max.emission 338 nm) is quenched about 28% on saturation with NAD+. Results of fluorometric titration of di-FA-GPDH with NAD+ suggest the presence of two tight and two loose coenzyme binding sites (Kdiss. 0.1 and 6.0 microM, respectively). Initial rates of the NAD(+)-dependent reaction of di-FA-GPDH with arsenate and phosphate and of mono-FA-GPDH with phosphate have been determined at varying coenzyme concentrations. The data suggest that binding of NAD+ at the tight sites does not activate the acyl group for its reaction with the acceptor (phosphate or arsenate). The group transfer reaction is dependent only on NAD+ binding to the loose sites, which carry the acyl group. The large difference in the NAD+ binding affinity to the two types of sites and their different effects on the group transfer reaction impart a sigmoidal shape to the rate versus [NAD+] plots. The sigmoidicity is abolished if the reactive SH groups at the unacylated sites are blocked by carboxymethylation.     

Interactions and spatial arrangement of spin-labeled NAD+ bound to glyceraldehyde-3-phosphate dehydrogenase. Comparison of EPR and X-ray modeling data

The spatial arrangement of coenzyme NAD+ in remote and adjacent binding sites in various stoichiometric complexes with tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was examined via EPR spectroscopy. An adenosine N6-15N,2H17 spin-labeled derivative of coenzyme NAD+ (SL-NAD+) was chemically synthesized for this work. The spectral simplifications and narrow line widths afforded by 15N and 2H substitution enabled experimental EPR spectra to be deconvoluted into their three component spectra: (a) unbound coenzyme, (b) bound coenzyme without adjacent site occupied, and (c) bound coenzyme with adjacent site occupied. Binding of SL-NAD+ in adjacent active centers of R axis-related subunits resulted in resolved dipolar interactions which characterized intersubunit distances. Binding to distant subunits related by the P and Q axes gave no dipolar interaction. Once the first NAD+ site was occupied, EPR spectra at various stoichiometries provided evidence for nonpreferential spatial binding of SL-NAD+ to the three unoccupied sites. EPR spectral simulations indicated a separation of 12.8 A for the unpaired electrons of spin label moieties of R axis-related coenzymes. Molecular modeling based on x-ray crystallographic data predicted 11-13 A. The angles and distance relating to interacting spin-labels were calculated from atomic coordinates based on molecular modeling of both anti-anti and anti-syn (adenine-ribose) conformations of SL-NAD+. Computer-generated line shapes indicated best agreement with experimental EPR results when the anti-anti geometry was employed. Comparison of EPR spectra from soluble and ammonium sulfate-precipitated enzymes indicated that the NAD+-binding domains are positioned equivalently in the two physical states. Since the observed dipolar line shapes are critically dependent on the distance and geometry relating to the interacting SL-NAD+, these data provide direct evidence for a high degree of conservation of quaternary structure of the enzyme in the hydrated crystalline state. Studies on the enzyme isolated from human erythrocytes also indicated a close correlation with the rabbit muscle enzyme in both the arrangement of NAD+-binding domains and negative cooperativity of coenzyme binding.     

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.     

Isolation and properties of glyceraldehyde-3-phosphate dehydrogenase from a sturgeon from the Caspian Sea and its interaction with spin-labeled NAD+ derivatives

Glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) was isolated from a sturgeon, Huso huso, from the Caspian Sea. It is closely related to the enzyme from a Pacific sturgeon, Acipenser transmontanus, with respect to amino acid composition, steady-state kinetics and coenzyme binding. The latter, as studied by means of a spin-labeled derivative of NAD+, is negatively cooperative exhibiting a Hill coefficient of 0.84 at 12 degrees C. Two derivatives of NAD+ spin-labeled at N6 or C8 of the adenine ring were found to be active coenzymes with maximum velocities reaching 35 or 45% of the value for NAD+ itself. When more than two equivalents of either spin-labeled NAD+ are bound to the enzyme spin-spin interactions are observed in the ESR spectra. Distances between the nitroxide radicals (8--9 A) calculated from the observed splittings are in excellent agreement with data predicted from the crystal structure of the lobster enzyme when the coenzyme is bound in an anti-conformation of the adenine moiety about the glycosidic bond to all four subunits.     

Inhibition of the methylcoenzyme M methylreductase system by NAD+ and NADP+ in cell-extracts of Methanosarcina barkeri

In cell extracts of Methanosarcina barkeri, the methylcoenzyme M methylreductase system with H2 as the electron donor was inhibited by NAD+ and NADP+, but NADH and NADPH had no effect on enzyme activity. NAD+ (4 and 8 mM) shifted the saturation curve for methylcoenzyme M from hyperbolic (Hill coefficient [nH] = 1.0; concentration of substrate giving half maximal velocity [Km] = 0.21 mM) to sigmoidal (nH = 1.5 and 2.0), increased Km (Km = 0.25 and 0.34 mM), and slightly decreased Vmax. Similarly NADP+ at 4m and 8 mM increased nH to 1.6 and 1.85 respectively, but the Km values (0.3 and 0.56 mM) indicated that NADP+ was a more efficient inhibitor than NAD+.     

Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.     

UDP-galactose 4-epimerase. Phosphorus-31 nuclear magnetic resonance analysis of NAD+ and NADH bound at the active site

The phosphorus atoms of NAD+ bound within the active site of UDP-galactose 4-epimerase from Escherichia coli exhibit two NMR signals, one at delta = -9.60 +/- 0.05 ppm and one at delta = -12.15 +/- 0.01 ppm (mean +/- standard deviation of four experiments) relative to 85% H3PO4 as an external standard. Titration of epimerase.NAD+ with UMP causes a UMP-dependent alteration in the chemical shifts of the resulting exchange-averaged spectra, which extrapolate to delta = -10.51 ppm and delta = -11.06 ppm, respectively, for the fully liganded enzyme, with an interconversion rate between epimerase.NAD+ and epimerase.NAD+.UMP of at least 490 s-1. Conversely, the binding of 8-anilinonaphthalene-1-sulfonate, which is competitive with UMP, causes a significant sharpening of the epimerase.NAD+ resonances but very little alteration in their chemical shifts, to delta = -9.38 ppm and delta = -12.16 ppm, respectively. UMP-dependent reductive inactivation by glucose results in the convergence of the two resonances into a single signal of delta = -10.57 ppm, with an off-rate constant for UMP dissociation from the epimerase.NADH.UMP complex estimated at 8 s-1. Reductive inactivation by borohydride under anaerobic conditions yields a single, broad resonance centered at about delta = -10.2 ppm. The data are consistent with, and may reflect, the activation of NAD+ via a protein conformational change, which is known from chemical studies to be driven by uridine nucleotide binding. Incubation of epimerase.NAD+ with UMP in the absence of additional reducing agents causes a very slow reductive inactivation of the enzyme with an apparent pseudo-first-order rate constant of 0.013 +/- 0.001 h-1, which appears to be associated with liberation of inorganic phosphate from UMP.     

Coenzyme properties of NAD+ bound to different matrices through the amino group in the 6-position.

A method for the synthesis of N6-(2-aminoethyl)-NAD+ is given. The binding of this NAD+ derivative to different soluble and insoluble supports and the direct coupling of NAD+ to epoxyactivated Sepharose are described. Proofs are given that NAD+ is bound through the amino group in 6- position and the NAD+ derivative through the aliphatic amino group of the side chain. Non-enzymic reduction of the bound coenzyme to an almost quantitative extent is possible in all cases, but the enzymic reduction is largely influenced by the support. While N6-(2-aminoethyl)-NAD+ coupled to soluble dextran is nearly completely reducible by different dehydrogenases with a velocity of about 40% of that for free NAD+, the coenzyme bound to different insoluble matrices is very slowly reduced. Only 5% of the coenzyme derivative bound to BrCN-activated Sepharose are reducible, but 40% when it is bound through a spacer. From capacity determinations evidence is given that, even in this coenzyme gel, only those coenzyme molecules are useful in affinity chromatography which are on the surface of the gel grains; it is supposed that this may be due to the slow diffusion of an enzyme into the inner parts of an affinity gel.     

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.     

Enhancement of pyruvate productivity in Torulopsis glabrata: Increase of NAD+ availability

This study aimed at increasing the pyruvate productivity from a multi-vitamin auxotrophic yeast Torulopsis glabrata, by increasing the availability of NAD+. We examined two strategies for increasing availability of NAD+. To supplement nicotinic acid (NA), the precursor of NAD+; and to increase the activity of alcohol dehydrogenase integrating with addition acetaldehyde as exterior electron acceptor. The addition of 8 mg l(-1) NA to the fermentation medium resulted in a significant increase in the glucose consumption rate (48.4%) and the pyruvate concentration (29%). An ethanol-utilizing mutant WSH-13 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the alcohol dehydrogenase activity of the mutant WSH-13 increased about 110% and the mutant could utilize ethanol as the sole carbon source for growth (1.8 g l(-1) dry cell weight). When growing with glucose, the addition of 4 mg l(-1) acetaldehyde to the mutant WSH-13 culture broth led to a significant increase in the glucose consumption rate (26.3%) and pyruvate production (22.5%), but the ratio of NADH/NAD+ decreased to 0.22. Acetaldehyde did not affect the glucose and energy metabolism at high dissolved oxygen (DO) concentration. However, at lower DO concentration (20%), maintaining the acetaldehyde concentration in the mutant culture broth at 4 mg l(-1) caused an increased NAD+ concentration but a decreased NADH concentration. As a consequence, the pyruvate production rate, the pyruvate yield on glucose and the pyruvate concentration were 68, 44 and 45% higher, respectively, than the corresponding values of the control (without acetaldehyde). The strategy for increasing the glycolytic flux and the pyruvate productivity in T. glabrata by increasing the availability of NAD+ may provide an alternative approach to enhance the metabolites productivity in yeast.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

Covalent binding of an NAD+ analogue to horse liver alcohol dehydrogenase in a ternary complex with pyrazole

Examination of the model of the fixation site of the adenosine phosphate part of NAD+ on horse liver alcohol dehydrogenase led us to synthesize a NAD+ analogue N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]-NAD+ in order to alkylate the carboxylic acid group of Asp-273 and to convert the normally dissociable coenzyme into a permanently bound prosthetic group. This NAD+ analogue is coupled to the horse liver alcohol dehydrogenase in the ternary complex formed with pyrazole. In these conditions the degree of fixation varies between 0.4 and 0.58 coenzyme molecule/enzyme subunit molecule. The N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]NAD+ acts as a true prosthetic group which can be reduced and reoxidized by a coupled substrate reaction and the internal activity of this holoenzyme corresponds to the amount of analogue incorporated.     

Spatial arrangement of coenzyme and substrates bound to L-3-hydroxyacyl-CoA dehydrogenase as studied by spin-labeled analogues of NAD+ and CoA.

The synthesis of nitroxide spin-labeled derivatives of S-acetoacetyl-CoA, S-acetoacetylpantetheine, and S-acetoacetylcysteamine is described. These compounds are active substrates of L-3-hydroxyacyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35] exhibiting vmax values from 20% to 70% of S-acetoacetyl-CoA itself. S-Acetoacetylpantetheine and S-acetoacetylcysteamine form binary complexes with the enzyme and exhibit ESR spectra typical for immobilized nitroxides. In the case of spin-labeled pantetheine, the radical is more mobile. When spin-labeled substrates are bound simultaneously to each active site of this dimeric enzyme, spin-spin interactions differentiate between two alternate orientations of the substrate [Birktoft, J.J., Holden, H.M., Hamlin, R., Xuong, N.H., & Banaszak, L.J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8262-8266]. The fatty acid moiety is thought to be located in a cleft between two domains whereas a large part of the CoA moiety probably extends into the solution. NAD+, spin-labeled at N6 of the adenine ring, is an active coenzyme of L-3-hydroxyacyl-CoA dehydrogenase (60% vmax). Complexes with the enzyme exhibit ESR spectra typical of highly immobilized nitroxides. Binding of coenzyme NAD+ causes conformational changes of the binary enzyme/substrate complex as revealed by changes in the ESR spectrum of spin-labeled S-acetoacetylpantetheine.     

Binding of NAD+ to pertussis toxin.

The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a Kd value of 27 microM at 30 degrees C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ.mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule.     

NAD+ and NAD+ analogues in horse liver alcohol dehydrogenase. Relationship between reactivity and conformation simulated with molecular mechanics

In the present study we show that the enzymatic activity of the coenzyme nicotinamide adenine dinucleotide (NAD+) and its analogues (C(O)NH2 replaced by C(S)NH2, C(O)CH3, C(O)H and CN) with horse liver alcohol dehydrogenase (LADH) (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) can be rationalized by their conformation in the active site determined with molecular mechanics (AMBER, assisted model building with energy refinement). In order to establish the relation between the hydride transfer rate and the conformation of the NAD+ and its analogues, kinetic experiments with the poor substrate isopropanol were carried out. It appears that the enzymatic activity can be readily explained by the geometry of the pyridinium ring, in particular the magnitude of the 'out-of-plane' rotation of the carboxamide side chain (or analogues). The latter is nicely illustrated in the case of 3-cyanopyridine adenine dinucleotide which lacks any 'out-of-plane' rotation and concomitantly exhibits no significant enzymatic activity.     

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.     

Formation of transient complexes in the glutamate dehydrogenase catalyzed reaction.

The reaction of glutamate dehydrogenase and glutamate (gl) with NAD+ and NADP+ has been studied with stopped-flow techniques. The enzyme was in all experiments present in excess of the coenzyme. The results indicate that the ternary complex (E-NAD(P)H-kg) is present as an intermediate in the formation of the stable complex (E-NAD(P)H-gl). The identification of the complexes is based on their absorption spectra. The binding of the coenzyme to (E-gl) is the rate-limiting step in the formation of (E-NAD(P)H-kg) while the dissociation of alpha-ketoglutarate (kg) from this complex is the rate-limiting step in the formation of (E-NAD(P)H-gl). The Km for glutamate was 20-25 mM in the first reaction and 3 mM in the formation of the stable complex. The Km values were independent of the coenzyme. The reaction rates with NAD+ were approximately 50% greater than those with NADP+. Furthermore, high glutamate concentration inhibited the formation of (E-NADH-kg) while no substrate inhibition was found with NADP+ as coenzyme. ADP enhanced while GTP reduced the rate of (E-NAD(P)H-gl) formation. The rate of formation of (E-NAD(P)H-kg) was inhibited by ADP, while it increased at high glutamate concentration when small amounts of GTP were added. The results show that the higher activity found with NAD+ compared to NADP+ under steady-state assay conditions do not necessarily involve binding of NAD+ to the ADP activating site of the enzyme. Moreover, the substrate inhibition found at high glutamate concentration under steady-state assay condition is not due to the formation of (E-NAD(P)H-gl) as this complex is formed with Km of 3 mM glutamate, and the substrate inhibition is only significant at 20-30 times this concentration.