Altered cell shape is linked to increased p34cdc2 gene expression in fibroblasts expressing a mutant E2F-1 transcription factor

Transhydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.     

Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. Characterization of the beta H91, beta D392, and beta K424 mutants

Conserved and semiconserved acidic and basic residues of the beta subunit of the proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli potentially involved in proton pumping were investigated. Out of 16 charged residues studied, 6 have not been previously investigated. The most dramatic effects of mutation were observed with beta H91, beta D392, and beta K424. beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP+ by NADH (forward reaction) and reduction of 3-acetylpyridine-NAD+ by NADPH (reverse reaction) to lower pH. This mutant catalyzed a cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADP(H) with a pH profile also shifted toward a lower pH. These results are consistent with a mechanism where the normal forward and reverse reactions are indeed limited by protonation/deprotonation of beta H91. The cyclic reaction was affected by mutations of beta H91, probably through conformational changes involving the active NADP(H) site. The beta D392A mutant was inactive with regard to forward and reverse reactions, but showed a wild-type-like pH dependence for the partly active cyclic reaction. However, Km,app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site. Transhydrogenases contain a conserved beta K424-beta R425-beta S426 sequence that has been proposed to be important for NADP(H) binding. beta K424R was strongly inhibited and showed an 18-fold increased Km,app for NADPH in the reverse reaction as compared to wild type. Consequently, this mutation affected all NADP(H)-linked activities and essentially abolished the unspecific interaction of NAD(H) with this site. The pH dependences of the forward and reverse reactions, as well as the cyclic reaction, were shifted to a lower pH as compared to the wild-type enzyme, and the salt dependence was also altered.     

The E6 and E7 genes of human papilloma virus-type 16 protect primary astrocyte cultures from injury

Nicotinamide nucleotide transhydrogenase constitutes a proton pump which links the NAD(H) and NADP(H) pools in the cell by catalyzing a reversible reduction of NADP+ by NADH. The recent cloning and characterization of several proton-pumping transhydrogenases show that they share a number of features. They are composed of three domains, i.e., the hydrophilic domains I and III containing the NAD(H)- and NADP(H)-binding sites, respectively, and domain II containing the transmembrane and proton-conducting region. When expressed separately, the two hydrophilic domains interact directly and catalyze hydride transfer reactions similar to those catalyzed by the wild-type enzyme. An extensive mutagenesis program has established several amino acid residues as important for both catalysis and proton pumping. Conformational changes mediating the redox-driven proton pumping by the enzyme are being characterized. With the cloned, well-characterized and easily accessible transhydrogenases from E. coli and Rhodospirillum rubrum at hand, the overall aim of the transhydrogenase research, the understanding of the conformationally driven proton pumping mechanism, is within reach.     

Interactions of reduced and oxidized nicotinamide mononucleotide with wild-type and alphaD195E mutant proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli

The interaction of reduced nicotinamide mononucleotide (NMNH), constituting one half of NADH, with the wild-type and alphaD195E proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated. Reduction of thio-NADP+ by NMNH was catalysed at approximately 30% of the rate with NADH. Other activities including proton pumping and the cyclic reduction of 3'-acetyl-pyridine-NAD+ by NMNH in the presence of NADP+ were more strongly inhibited. The alphaD195 residue is assumed to interact with the 2'-OH moiety of the adenosine-5'-phosphate, i.e., the second nucleotide of NADH. Mutation of this residue to alphaD195E resulted in a 90% decrease in activity with NMNH as well as NADH as substrate, suggesting that it produced global structural changes of the NAD(H) binding site. The results suggest that the NMN moiety of NADH is a substrate of transhydrogenase, and that the adenine nucleotide is not required for catalysis or proton pumping.     

Positron-emission tomography with fluorine-18-deoxyglucose in the staging and control of patients with lymphoma. Comparison with clinico-radiologic assessment

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+ coupled to the translocation of protons across the cytoplasmic membrane. It is composed of two subunits (alpha, beta) organized as an alpha 2 beta 2 tetramer. This brief review describes the use of site-directed mutagenesis to investigate the structure, mechanism and assembly of the transhydrogenase. This technique has located the binding sites for NAD(H) and NADP(H) in the alpha and beta subunits, respectively. Mutagenesis has shown that the cysteine residues of the enzyme are not essential for its function, and that inhibition of the enzyme by sulfhydryl-specific reagents must be due to perturbation of the three-dimensional structure. The sites of reaction of the inhibitors N,N'-dicyclohexylcarbodiimide and N-(1-pyrene)maleimide have been located. Selective mutation and insertion of cysteine residues followed by cupric o-phenanthrolinate-induced disulfide crosslinking has defined a region of interaction between the alpha subunits in the holoenzyme. Determination of the accessibility of selectively inserted cysteine residues has been used to determine the folding pattern of the transmembrane helices of the beta subunit. Site-directed mutagenesis of the transmembrane domain of the beta subunit has permitted the identification of histidine, aspartic acid and asparagine residues which are part of the proton-pumping pathway of the transhydrogenase. Site-directed mutagenesis and amino acid deletions have shown that the six carboxy terminal residues of the alpha subunit and the two carboxy terminal residues of the beta subunit are necessary for correct assembly of the transhydrogenase in the cytoplasmic membrane.     

NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax. The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway.     

Reaction of the NAD(P)H:flavin oxidoreductase from Escherichia coli with NADPH and riboflavin: identification of intermediates

Flavin reductase catalyzes the reduction of free flavins by NAD(P)H. As isolated, Escherichia coli flavin reductase does not contain any flavin prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin substrate in a ternary complex prior to oxidoreduction. The reduction of riboflavin by NADPH catalyzed by flavin reductase has been studied by static and rapid kinetics absorption spectroscopies. Static absorption spectroscopy experiments revealed that, in the presence of riboflavin and reduced pyridine nucleotide, flavin reductase stabilizes, although to a small extent, a charge-transfer complex of NADP+ and reduced riboflavin. In addition, reduction of riboflavin was found to be essentially irreversible. Rapid kinetics absorption spectroscopy studies demonstrated the occurrence of two intermediates with long-wavelength absorption during the catalytic cycle. Such intermediate species exhibit spectroscopic properties similar to those of charge-transfer complexes of oxidized flavin and NAD(P)H, and reduced flavin and NAD(P)+, respectively, which have been identified as intermediates during the reaction of flavoenzymes of the ferredoxin-NADP+ reductase family. Thus, a minimal kinetic scheme for the reaction of flavin reductase with NADPH and riboflavin can be proposed. After formation of the Michaelis complex of flavin reductase with NADPH and riboflavin, a first intermediate, identified as a charge-transfer complex of NADPH and riboflavin, is formed. It is followed by a second charge-transfer intermediate of enzyme-bound NADP+ and reduced riboflavin. The latter decays, yielding the Michaelis complex of flavin reductase with NADP+ and reduced riboflavin, which then dissociates to complete the reaction. These results support the initial hypothesis of a structural similarity between flavin reductase and the enzymes of the ferredoxin-NADP+ reductase family and extend it at a functional level.     

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures

Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by alpha-oxidation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate:acceptor oxidoreductase was purified and studied. The oxygen-sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an alpha2 beta2 gamma2 delta2 epsilon2 composition. Phenylglyoxylate:acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 microM) and coenzyme A (Km 55 microM); 2-oxoisovalerate was oxidized with 15% of the rate. The turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H:viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha2 beta2 gamma2 delta2, as reported for archaeal 2-oxoacid:ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an epsilon2 unit that catalyzes the transfer of electrons from a small ferredoxin-like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin:NAD(P)+-oxidoreductase.     

Aspartate dehydrogenase in vitamin B12-producing Klebsiella pneumoniae IFO 13541

We found a new reaction of aspartic acid dehydrogenation, catalyzed by NADP(+)-dependent aspartate dehydrogenase, in vitamin B12-producing Klebsiella pneumoniae IFO 13541. The enzyme, which was purified from a crude extract of K.pneumoniae IFO 13541, catalyzes the oxidative deamination of aspartic acid to form oxaloacetic acid. This enzyme had a molecular mass of about 124 kDa consisting of two identical subunits. L-Aspartic acid was a substrate, although D-aspartic acid and L-glutamic acid were inactive. The enzyme showed maximal activity at about pH 7.0-8.0 for the oxidative deamination of L-aspartic acid, and it required NADP+ as a coenzyme, while NAD+ was inactive.     

Potential difference across subcellular particle membranes. II. Compensation method of measurement

"Zero-loop" of the molecular potential transformer of submitochondrial particles (SMP) is separated from the remaining electron transfer chain by rotenone, and its e.m.f. ET=0,003+RT/2F in [NADP X H] [NAD+]/[NADP+] [NAD X H] volts is used in the compensative method of measurement of the potential difference across the SMP membrane (delta USMP). The phospholipid membrane, measuring the concentration of the penetrating anions in the solution contained SMP, is used as "zero-indicators". This concentration drops monotonically with increase in delta USMP. Delta USMP is equal to ET when the addition of substrates of transhydrogenase reaction with definite ET does not change the potential across phospholipid membrane.     

A membrane-bound NAD(P)+-reducing hydrogenase provides reduced pyridine nucleotides during citrate fermentation by Klebsiella pneumoniae

During anaerobic growth of Klebsiella pneumoniae on citrate, 9.4 mmol of H2/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO2. Upon addition of NiCl2 (36 microM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H2-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)+ was also achieved by the addition of formate. In crude extracts, the H2:NAD+ oxidoreductase activity was 0.13 micromol min-1 mg-1, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 micromol of NADPH formed min-1 mg-1 at pH 7.0 and 1.7 micromol of NADH formed min-1 mg-1 at pH 9.5. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the Na+/H+ antiporter monensin, the H2-dependent reduction of NAD+ by membrane vesicles decreased only slightly (about 16%). The NADP+- or NAD+-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-N-oxide or Triton X-100. NAD(P)H formation with H2 as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by K. pneumoniae during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H2:NAD(P)+ oxidoreductase that provides reducing equivalents for the synthesis of cell material.     

Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species

Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.     

Fluorescence microscopic observation of catalysis by single or few LDH-1 enzyme molecules

Lactate dehydrogenase (LDH-1) catalyzes the reaction of lactate and nonfluorescent NAD+ to pyruvate, NADH (fluorescence at lambda em = 455 nm, lambda em = 365 nm) and H+. The injection of highly diluted LDH-1 solution into a drop of substrate solution results in the formation of a bubble of enzyme inside the drop of substrate. At the contact surface between the enzyme solution and the substrate, discrete and statistically distributed zones of increasing fluorescence intensity and different size can be observed after enzyme injection. These zones can be interpreted as clouds of NADH around a single or a few enzyme molecules. The kinetics of the NADH formation in every fluorescent zone, and the size of the zone, can be described by a zero order production combined with a diffusion controlled loss of the reaction's product NADH from the reaction zone. From the dilution of the enzyme solution and from statistical analysis one can conclude that only few enzyme molecules in the center of the fluorescent reaction zones catalyze the NADH formation.     

Re-face stereospecificity at C4 of NAD(P) for alcohol dehydrogenase from Methanogenium organophilum and for (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as determined by 1H-NMR spectroscopy

The two diastereotopic protons at C4 of NAD(P)H are seen separately in 1H-NMR spectra. This fact was used to determine the stereospecificity at C4 of NAD(P) for the NADP-dependent alcohol dehydrogenase from Methanogenium organophilum and for the NAD-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans. The reduction of NADP+ with [2H6]ethanol was found to yield (4R)-[4-2H1]NADPH and the oxidation of (4R)-[4-2H1]NADH with 2-oxoglutarate to yield unlabelled [4-1H]NAD+. These results indicate that both enzymes are Re-face stereospecific at C4 of the pyridine nucleotides.     

Cartilage degeneration in relation to repetitive pressure: case study of a unilateral hip hemiarthroplasty patient

The mitochondrial, inner-membrane-associated, reversible NADPH-->NAD transhydrogenase of adult Hymenolepis diminuta physiologically couples matrix-localized, NADP-specific "malic" enzyme with NADH-dependent anaerobic electron transport. Employing submitochondrial particles (SMP) as the source of enzyme activity and both spectrophotometric and fluorometric assessments, the present study made evident that in its catalysis of transhydrogenation between NADPH and NAD, the cestode enzyme engages in the concomitant transmembrane translocation of protons. As assessed spectrophotometrically, the catalysis of NADPH-dependent NAD reduction by H. diminuta SMP was stimulated significantly by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), as well as by the protonophoric anthelmintic, niclosamide. In addition, N,N'-dicyclohexylcarbodiimide (DCCD) markedly diminished SMP-catalyzed hydride ion transfer between NADPH and NAD. The catalysis by SMP of concomitant, transhydrogenase-mediated proton translocation was evaluated more directly via fluorometric assays using 8-anilino-1-napthalenesulfonic acid (ANS) as the probe. These latter evaluations revealed a transhydrogenase-dependent enhancement of ANS fluorescence in accord with an intravesicular accumulation of protons. ANS fluorescence was quenched rapidly when the assay system was supplemented with CCCP, FCCP, or niclosamide. Consistent with the helminth transhydrogenase acting as a proton pump, transhydrogenase-mediated enhanced fluorescence also was inhibited by DCCD. Considered collectively, these data indicated, apparently for the first time for any invertebrate system, that the transhydrogenase, in catalyzing the NADPH-->NAD reaction, acts in the translocation of protons from the matrix to the intermembrane space mitochondrial compartment.     

Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.     

Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli

A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.     

The conformation of NADH bound to inosine 5'-monophosphate dehydrogenase determined by transferred nuclear Overhauser effect spectroscopy

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The reaction proceeds with concomitant conversion of NAD+ to NADH and is the rate-limiting step in the de novo biosynthesis of guanosine nucleotides. IMPDH is a target for numerous chemotherapeutic agents. The conformations of enzyme-bound substrates, enzyme-bound products and enzyme-bound ligands in general, are of interest for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. Although several of the chemotherapeutic inhibitors of IMPDH are NAD+ or NADH analogues, no structural data for IMPDH-bound NAD+ (or NADH) are available. In the present work, we have used transferred nuclear Overhauser effect spectroscopy (TRNOESY) to determine the conformation of NADH bound to the active site of human type II IMPDH (IMPDH-h2). The inter-proton distances determined from TRNOESY data indicate that NADH binds to the enzyme active site in an overall extended conformation. The adenosine moiety and the nicotinamide riboside moiety are both in the anti conformation about the glycosidic bond, and both ribose rings are in approximately C4'-exo conformations. The nicotinamide amide group was found to be in a cis conformation. The anti conformation of the nicotinamide riboside moiety is in accord with the preferred conformations of several potent and selective dinucleotide inhibitors and is consistent with that implied by the stereospecificity of hydride transfer in the enzymatic reaction. The implications of this conformation for the catalytic mechanism of IMPDH-h2 are discussed.     

Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase

Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics. Initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism. NADH oxidation proceeds stereospecifically with the transfer of the pro-R hydrogen to the enzyme and the amide moiety of the nicotinamide appears to be the principal mediator of the interaction between NR and NADH. 2,4-Dinitrotoluene is the most efficient oxidizing substrate examined, with a kcat/KM an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a Ki of 62 nM. Several compounds containing a carboxyl group are also competitive inhibitors vs. NADH. Yonetani-Theorell analysis of dicoumarol and acetate inhibition indicates that their binding is mutually exclusive, which suggests that the two inhibitors bind to the same site on the enzyme. NAD+ does not exhibit product inhibition and in the absence of an electron acceptor, no isotope exchange between NADH and 32P-NAD+ could be detected. NR catalyzes the 4-electron reduction of nitrobenzene to hydroxylaminobenzene with no optically detectable net formation of the putative two-electron intermediate nitrosobenzene.     

Hyperprolactinemia in males with systemic lupus erythematosus

Alanine dehydrogenase [EC 1. 4. 1. 1] was purified to homogeneity from a crude extract of Enterobacter aerogenes ICR 0220. The enzyme had a molecular mass of about 245 kDa and consisted of six identical subunits. The enzyme showed maximal activity at about pH 10.9 for the deamination of L-alanine and at about pH 8.7 for the amination of pyruvate. The enzyme required NAD+ as a coenzyme. Analogs of NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide served as coenzymes. Initial-velocity and product inhibition studies suggested that the deamination of L-alanine proceeded through a sequential ordered binary-ternary mechanism. NAD+ bound first to the enzyme, followed by L-alanine, and the products were released in the order of ammonia, pyruvate, and NADH. The Km were 0.47 mM for L-alanine, 0.16 mM for NAD+, 0.22 mM for pyruvate, 0.067 mM for NADH, and 66.7 mM for ammonia. The Km for L-alanine was the smallest in the alanine dehydrogenases studied so far. The enzyme gene was cloned into Escherichia coli JM109 cells and the nucleotides were sequenced. The deduced amino acid sequence was very similar to that of the alanine dehydrogenase from Bacillus subtilis. However, the Enterobacter enzyme has no cysteine residue. In this respect, the Enterobacter enzyme is different from other alanine dehydrogenases.