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1.
Mark A. Jensen  Philip J. Elving 《BBA》1984,764(3):310-315
The rate constant, kd, for the dimerization of the free radical (NAD·), produced on the initial one-electron reduction of NAD+, was measured by double potential-step chronoamperometry, fast-scan cyclic voltammetry (cathodic-anodic peak current ratio) and slow-scan cyclic voltammetry (peak potential shift) for a medium in which neither NAD+ nor its reduction products are adsorbed at the solution/electrode interface. All three methods give concordant values of kd (approx. 3·107 M?1·s?1), which are in reasonable accord with the values determined by pulse radiolysis but are considerably greater than values previously determined electrochemically. For the NAD+/NAD· couple, the heterogeneous rate constant (ks,h) exceeds 1 cm·s?1 at 25°C and the formal potential (E0c) vs. sce is ? 1.155 V at 25°C and ? 1.149 V at 1°C at pH 9.1, with an uncertainty of about ±0.005 V.  相似文献   

2.
Metabolism of γ-Aminobutyrate in Agaricus bisporus. III. The Succinate-Semialdehyde: NAD (P)+ Oxidoreductase. The succinate-semialdehyde:NAD(P)+ oxidoreductase (E.C. 1.2.1.16) is responsible for the second step in the catabolism of γ-aminobutyrate: the irreversible enzymatic conversion of succinic semialdehyde (SSA) to succinate. Succinate semialdehyde dehydrogenase was extracted from mitochondrial fraction of fruit-bodies of Agaricus bisporus Lge. The mitochondrial pellet was sonicated and centrifuged at 110,000 g; the supernatant obtained was designated the “crude extract”. The enzyme was extremely unstable on storage, unless 1 mM EDTA and 20% glycerol were added. Kinetic studies were carried out at 30°C, and the formation of NADH or NADPH was followed by measuring increase of absorbance at 340 nm with a spectrophotometer. The dehydrogenase was completely inactive when the reaction was run in the absence of thiol and was more active with NAD+ than with NADP+. In the “crude extract” the activity with NADP+ had a pH optimum between 8.6 and 9.1 and the Km values for SSA and NADP+ were 2.0 × 10?4M and 1.4 × 10?4M respectively. The pH optimum with NAD+ was found between 8.6 and 8.8 and the Km value for SSA is 4.8 × 10?4M and for NAD+ 2.0 × 10?3M. With NAD+, the kinetic values (pH, Km) of the “crude extract” chromatographed on hydroxylapatite were unchanged. Inhibition by thiamine pyrophosphate (TPP) was uncompetitive with respect to NAD+, those by malate, ATP, ADP and NADPH non-competitive and that by NADH competitive. These results and the fact that activity with NAD+ was lost more slowly than with NADP+ indicate the possibility of at least two mitochondrial succinate-semialdehyde dehydrogenases, even though the activities of this enzyme assayed with NAD+ and NADP+ respectively were not able to be separated from each other by hydroxylapatite column chromatography. Some speculations on the metabolic regulation of this dehydrogenase and considerations on the significance of these results in the physiology of respiration in Agaricus bisporus Lge are given.  相似文献   

3.
Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD+ as a cofactor. The recombinant caXDH has a KM of 8.8 mM and 37.7 μM using the substrate xylitol and NAD+, respectively, and its catalytic efficiency is 53,200 min?1 mM?1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD+ to NADP+ in which the KM and kcat/KM towards NADP+ are 119 μM and 26,200 min?1 mM?1, respectively.  相似文献   

4.
The typical procedure for determining NAD+-malic enzyme (EC 1.1.1.39) is to calculate the enzyme rate to be ΔA340/Δ time after the endogenous NAD+-malic dehydrogenase (EC 1.1.1.37) catalyzed reaction has reached equilibrium. This ignores the equilibrium shift of oxaloacetic acid and NADH during the course of the NAD+-malic enzyme reaction and causes an error that varies depending on the reagent [malate], [NAD+], pH and final [NADH]. For a ΔA340 of 0.02, the error is about 80% and for a ΔA340 of 0.30, 20%. We develop this argument, give supportive data and present a simple method to circumvent the error.  相似文献   

5.
The use of 4,4′-bis-dimethylaminodiphenylcarbinol (BDC-OH) as an analytical reagent for sulfhydryl residues and as a specific chemical modification reagent for proteins is dependent upon the unique properties of the BDC+ cation present in aqueous buffers below a pH of 6.5. In the presence of aqueous buffers, pH 5.1, BDC+ exhibits a λmax of 606 nm with an apparent molar absorption coefficient of 10,000 m?1 cm?1. Upon the addition of 4m guanidine hydrochloride this apparent coefficient is enhanced to 70,800 m?1 cm?1. The true molar extinction coefficient for BDC+ was determined to be 128,000 m?1 cm?1. The reaction of BDC+ with sulfhydryl residues of proteins or simple thiols is rapid and leads to a complex devoid of visible color. In the pH range 3.0–7.0, a complex equilibrium is established among the three species BDC-OH, BDC+, BDCH++. The formation of this equilibrium is proton mediated, and is discussed in terms of the equilibrium, rate, and acid dissociation constants.  相似文献   

6.
Membrane potential, Vm, and K+(86Rb+) fluxes have been measured simultaneously on individual cells of Acetabularia mediterranea. During resting state (resting potential approx. ?170 mV) the K+ influx amounts to 0.24–0.6 pmol · cm?2 · s?1 and the K+ efflux to 0.2–1.5 pmol · cm?2 s?1. According to the K+ concentrations inside and outside the cell (40 : 1) the voltage dependent K+ flux (zero at Vm = EK = ?90 mV) is stimulated approx. 40-fold for Vm more positive than EK.It is calculated that during one action potential (temporary depolarization to Vm more positive than EK) a cell looses the same amount of K+, which leaks in during 10–20 min in the resting state (Vm = ?170 mV). Since action potentials occur spontaneously in Acetabularia, they are therefore suggested to have a significant function for the K+ balance of this alga.  相似文献   

7.
The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH3 and the nicotinamide ring of αNAD+ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD+ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD+.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10?3 min?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M4 and H4 lactate dehydrogenase, are shown to oxidize αNADH to αNAD+ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD+in vivo.  相似文献   

8.
Coherent anti-Stokes Raman scattering spectra, in resonance with the isoalloxazine visible electronic transition, have been obtained down to 300 cm?1 for flavin adenine dinucleotide, riboflavin binding protein and glucose oxidase, in H2O and D2O. Several isoalloxazine vibrational modes can be identified by analogy with those of uracil. Of particular interest is a band at ~1255 cm?1 in H2O, which is replaced by another at ~1295 cm?1, in D2O. The H2O band appears to be a sensitive monitor of H-bonding of the N3 isoalloxazine proton to a protein acceptor group. It shifts down by 10 cm?1 in riboflavin binding protein, and disappears altogether in glucose oxidase. Other band shifts, of 3–5 cm?1, are similar for the two flavoproteins, and may reflect environmental changes between aqueous solution and the protein binding pockets.  相似文献   

9.
It has been shown that direct excitation of NADH (or NADPH) in aqueous medium at 254 nm, or at wavelengths longer than 320 nm (where only the reduced nicotinamide moiety absorbs), leads to generation of NAD+ (or NADP+). The reaction proceeds both in the presence and absence of oxygen. Under aerobic conditions the reaction is accompanied by formation of H2O2 at a level equimolar with that of the NADH present in solution. On irradiation at wavelengths longer than 320 nm, conversion of NADH to enzymatically active NAD+ is about 75%. Under analogous irradiation conditions, the dimers (NAD)2 and (NADP)2 undergo disproportionation to NAD+ and NADP+, respectively, to the extent of 90%. Both physicochemical and enzymatic criteria were employed to formulate mechanisms for the photooxidation of NADH and the photodisproportionation of the dimer (NAD)2.  相似文献   

10.
Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K M of 99.0 μM (LpNox1) and 27.6 μM (LpNox2), and yielding catalytic efficiency k cat/K M of 1.0 and 0.2 μM?1 s?1, respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD+ was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD+ regeneration in dehydrogenase-catalyzed oxidations.  相似文献   

11.
The need for cost‐effective and sustainable power supplies has spurred a growing interest in hybrid energy harvesting systems, and the most elementary energy production process relies on intermittent solar power. Here, it is shown how the ambient mechanical energy leads to water splitting in a photoelectrochemical (PEC) cell boosted by a triboelectric nanogenerator (TENG). In this strategy, a flexible TENG collects and transforms mechanical energy into electric current, which boosts the PEC water splitting via the charged Li‐ion battery. Au nanoparticles are deposited on TiO2 nanoarrays for extending the available light spectrum to visible part by surface plasmon resonance effect, which yields a photocurrent density of 1.32 mA cm?2 under AM 1.5 G illumination and 0.12 mA cm?2 under visible light with a bias of 0.5 V. The TENG‐charged battery boosts the water splitting performance through coupling electrolysis and enhanced electron–hole separation efficiency. The hybrid cell exhibits an instantaneous current more than 9 mA with a working electrode area of 0.3 cm2, suggesting a simple but efficient route for simultaneously converting solar radiation and mechanical energy into hydrogen.  相似文献   

12.
A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of Mr 32,000 each was discernible. Km values for NAD+ and myo-inositol 1-phosphate have been recorded as 2.8 × 10?4 and 5.0 × 10?4m, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD+ are reduced.  相似文献   

13.
R.M. Simmons 《FEBS letters》1983,164(1):201-204
On incubation of B. subtilis RM125(arg15 leuA8 rM? mM?) with DNA from alkalophilic Bacillus, the transformants (Arg+Leu? or Leu?Arg+) appeared at pH 10. The transformants were able to grow even at pH 7. Alkalophilic Bacillus was resistant to bacteriophages π105D1C2·1012 grown on B. subtilis 1012(r-mM+) and π105D1C2·ISMR4 grown on B. subtilis ISMR4rM+rR+mM+mR+), but the recipient B. subtilis and the transformant(Arg+Leu?) were susceptible to both the of the bacteriophages. The results indicate that the transformant is a B. subtilis derivative and that alkalophilicity of alkalophilic Bacillus was transferred to B. subtilis.  相似文献   

14.
4,4′-bis-Dimethylaminodiphenylcarbinol (BDC-OH) dissociates in aqueous buffers at pH values below neutrality to form a resonance-stabilized carbonium-immonium ion (BDC+) which exhibits an absorbance maximum at 606 nm. In the presence of 4.0 M guanidine hydrochloride, BDC+ has an apparent molar absorption coefficient of 70,800 M?1cm?1 and an absorbance maximum of 612 nm. Sulfhydryl groups react with the cation to form S-(4,4′-bis-dimethylaminodiphenylmethyl-) derivatives with a concomitant quantitative loss of the 612-nm absorbance. This quantitative interaction has been exploited in the development of a new and convenient technique for the quantitative determination of sulfhydryl groups in proteins. Results of sulfhydryl determinations on simple thiols and five proteins are presented, along with comparison data obtained via other sulfhydryl techniques.  相似文献   

15.
Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo‐ and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the cofactor binding site seems crucial in determining cofactor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2′‐phosphate group of NADP(H). Consistent with crystal structures, steady state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (kcat/KM) of NADP+ at least an order of magnitude lower than NAD+. Substitution of Glu42 with Ala improved specificity toward NADP+ by 10‐fold compared to wild type. The cofactor bound structure uncovered a novel nicotinamide‐hydroxyl ion (NAD+‐OH?) adduct contraposing previously reported adducts. The OH? of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2′‐hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations, we propose an involvement of NAD+‐OH? adduct in proton relay instead of hydride transfer as noted for previous adducts. Proteins 2014; 82:216–229. © 2013 Wiley Periodicals, Inc.  相似文献   

16.
The thiolytic cleavage of O-2,4-dinitrophenyl (Dnp) derivatives of phenols was applied to the synthesis of tyrosine-containing peptides. This paper describes the preparation and properties of starting materials for such syntheses and illustrates their use in the synthesis of some peptides containing tyrosine at either the C- or N-terminus. A spectrophotometric method for following the thiolytic removal of Dnp groups from O-Dnp-tyrosines was developed and used to establish optimal conditions for quantitative deblockage in aqueous and nonaqueous solvents. The method is based on the fact that upon thiolysis, the colorless solution of O-Dnp-tyrosine (λmax at 298 nm, pH 8.5) becomes yellow due to the formation of a dinitrophenylated thiol (for S-Dnp-2-mercaptoethanol, λmax at 340 nm, pH 8.5). This gives rise to a difference spectrum with a maximum at 354 nm (Δ?M = + 8680 M?1 cm?1), a minimum at 298 nm (Δ?M = ?5900 M?1 cm?1) and a crossover point at 318 nm, which is different (in the 290–320 nm range) from the difference spectrum obtained upon thiolysis of NIm-Dnp-histidine. This method provides a useful analytical tool in peptide and polypeptide synthesis as well as in protein chemistry.  相似文献   

17.
Quasi-elastic light scattering has been used to characterize the oligomeric properties of solutions of glycerol-cycled bovine microtubule protein, and the properties of the 30 S oligomeric species and 6 S tubulin heterodimer prepared by gel filtration on Sepharose 6B. It is shown that in dimer preparations, as little as 0.04% by number of 30 S rings would account for the difference between an observed mean diffusion coefficient D20, W = 3.1 × 10?7 cm2 s?1 and the value of D20, W = 5.1 × 10?7 cm2 s?1 calculated for tubulin dimer of Mrel 100,000. The 30 S ring has an observed diffusion coefficient of D20, W = 0.49 × 10?7 cm2 s?1. These values are not changed significantly by the presence of 4 m-glycerol, indicating the persistence of 6 S and 30 S forms for dimer and ring, respectively.Mixtures of ring and dimer components of this preparation behave as a non-interacting two-component system, indicating the absence of substantial re-equilibration between the species at 5 °C and pH 6.5.The effect of salt on ring and microtubule protein samples indicates partial dissociation, consistent with the formation of additional intermediate oligomeric forms.In quasi-elastic light scattering measurements adapted to kinetic studies, changes in the oligomeric composition of microtubule protein are detected in the early stages of the reversible assembly process at pH 6.5. A 25% decrease in scattered light intensity, without significant change in mean diffusion coefficient, indicates the lability of the ring oligomeric structures, which undergo partial transformation to alternative oligomeric species under these assembly conditions.  相似文献   

18.
ADP-ribosyl cyclase and NAD+ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD+, nicotinamide adenine dinucleotide phosphate (NADP+) or nicotinamide mononucleotide (NMN+) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (kcat/KM = 0.9-10 μM−1 s−1) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the kcat/KM values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM−1 s−1). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN+, kcat/KM = 0.05-0.4 μM−1 s−1. The kcat/KM values for the exchange reaction with NMN+ were generally similar (0.04-0.12 μM−1 s−1) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The kcat/KM values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.  相似文献   

19.
Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP+ (k cat/K m = 2618 mM?1 s?1, k cat = 249 s?1, K m = 0.10 ± 0.01 mM) as cofactor, although NAD+ (k cat/K m = 138 mM?1 s?1, k cat = 604 s?1, K m = 4.37 ± 0.56 mM) could also be accepted. The K m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP+ and NAD+ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP+ and at pH 7.0–8.6 for NAD+ while the optimal temperature was 80 °C for NADP+ and 70 °C for NAD+. This was the first observation that the NADP+-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD+-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.  相似文献   

20.
Addition of NAD+ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O2 uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD+ (NAP4-NAD+), an inhibitor of NAD+ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD+-stimulated malate-cytochrome c reductase activity, and reduction of added NAD+ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD+ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD+ by storing on ice for 72 hours, was completely dependent on added NAD+, and the effect of NAD+ on these mitochondria was prevented by incubating them with NAP4-NAD+. External NAD+ reduction by these mitochondria was not affected by NAP4-NAD+. We conclude that all effects of exogenous NAD+ on plant mitochondrial respiration can be attributed to net uptake of the NAD+ into the matrix space.  相似文献   

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