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1.
Sinichi Nagata Richard Feicht Winfried Bette Helmut Günther Helmut Simon 《Applied microbiology and biotechnology》1987,26(3):263-267
Summary From a thermophilic bacillus a viologen dependent pyridine nucleotide oxidoreductase has been purified and partially characterized. Its apparent molecular weight is about 120000 consisting of two subunits of equal or very similar molecular weight. Per molecular weight of 120000 the enzyme contains 4 FAD. FMN or labile sulfur could not be detected. The physiological role of the enzyme is not clear. It reduces NAD as well as NADP at the expense of reduced methylviologen. The reduced pyridine nucleotides can be reoxidized with carbamoylmethylviologen. The seven determined K
m- and six K
i-values show that the enzyme is suitable for the regeneration of NADH, NADPH, NAD and NADP. The stability in presence of oxidized and reduced methylviologen at 35°C or 60°C is satisfying for preparative work.Abbreviations MV
Methylviologen species
- MV2+
1,1-dimethyl-4,4-bipyridinium dication=methylviologen oxidized
- MV-
methylviologen cation radical=methylviologen reduced
- CAV
carbamoylmethylviologen species
- VDPNOR
Viologen dependent pyridine nucleotide oxidoreductase
- DSM
Deutsche Sammlung für Mikroorganismen
- Tris
tris(hydroxymethyl)aminomethane 相似文献
2.
Johann Bader Helmut Günther Shinichi Nagata Hans-Jürgen Schuetz Marie-Luise Link Helmut Simon 《Journal of biotechnology》1984,1(2):95-109
Several yeasts, as well as aerobic and anaerobic bacteria catalyze the reduction of NAD and NADP in the presence of reduced methylviologen. The rates are usually much higher than those of reductions of unsaturated substrates by the organisms in cofermentations with carbohydrates. Since methylviologen can be continuously reduced at the cathode of an electrochemical cell it acts in catalytic amounts as a regenerable electron donor. Such systems may be superior to that with glucose as electron donor, because the NAD(P)H can be used exclusively for the reduction of the unsaturated substrate. The rate of the NAD(P)H formation depends very much on the organism and for the same organism on the growth procedure, the growth medium, the pretreatment of the cells, the pH, the buffer as well as on the ionic strength. Cells of Candida utilis which were frozen and thawed several times were superior to cells freshly harvested. Crude extracts revealed the best activities.Clostridia show the highest activities (up to 15 U per mg protein in the crude extract) and are suitable catalysts for the preparation of [4S-2H]NADH and [4S-2H]NADPH using 2H2O-buffer in an electrochemical cell.The combinations of Alcaligenes eutrophus or Clostridium kluyveri and Candida utilis extracts in the presence of methylviologen are effective systems to reduce hydroxyacetone with hydrogen gas as electron donor or in an electrochemical cell. In this combination of microorganisms NADH is formed mainly by A. eutrophus or C. kluyveri and consumed for the reduction of hydroxyacetone by a reductase present in Candida utilis. The productivity numbers of such combinations are 10–30 times higher than those of yeasts alone.NAD(P)H regeneration, methylviologen-dependent NAD(P)H formation, deuterated NAD(P)H, Clostridia, yeast, bioreduction 相似文献
3.
The use of pig heart dihydrolipoamide dehydrogenase (diaphorase) for the regeneration of NADH or NAD
Summary Incubations of freshly dissolved diaphorase with reduced methylviologen show in the first hours variable but usually rather low activities for the reduction of NAD with reduced methylviologen, as compared to lipoamide dehydrogenase activity at the expense of NADH. However, the former activity increases in a few hours by factors of 5–10 and is then very stable under operational conditions. The half life in the presence of reduced methylviologen at 35°C is >40 days, whereas the lipoamide dehydrogenase activity has a half life of only about 4 h. Even in a rigorously stirred electro-chemical cell the methylviologen dependent NAD reductase activity is very stable.The enzyme is also suitable for the regeneration of NAD if carbamoylmethylviologen is used. This mediator has a 145 mV less negative redox potential than methylviologen. Again the stability of the enzyme is rather high. Under operational conditions the activity increases for about 9 days and then stays constant for at least 11 days. 相似文献
4.
A chloroplast fraction from Chlamydomonas reinhardii cells can oxidize NADH in the light, unlike chloroplasts of higher plants. The Chlamydomonas preparation catalyzes electron flow from NADH to methylviologen or ferredoxin to evolve hydrogen (in the presence of a hydrogenase) or take up oxygen. The NADH photooxidation is sensitive to rotenone, dibromothymoquinone and dicyclohexylcarbodiimide. This suggests that a rotenone sensitive NADH dehydrogenase is coupled on the plastoquinone reduction site of the potosynthetic electron flow system. On sonication of the particles NADH photooxidation is lost but may be restored by a protein fraction from an acetone extract plus plastocyanin.Abbreviations DAD
diaminodurene
- DCCD
dicyclohexylcarbodiimide
- DCMU
(3,3-dichlorphenyl)-N·N dimethyl urea
- DBMIB
dibromothymoquinone
- DNP-INT
dinitro-phenylether of 2-iodo-4-nitrothymol
- MV
methylviologen
- chl
chlorophyll
Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday 相似文献
5.
A neutral flavin semiquinone species was formed upon photoreduction of Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase whereas no flavin radical was detected by anaerobic reduction with NADH in the presence of m-hydroxybenzoate. In the latter case, the formation of flavin semiquinone is apparently thermodynamically unfavorable. A stereospecificity for the abstraction of the 4R-position hydrogen of NADH has been demonstrated for this hydroxylase. Deuterium and tritium isotope effects were observed with (4R)-[4-2H]NADH and (4R)-[4-3H]NADH as substrates. The DV effect indicates the existence of at least one slow step after the isotope-sensitive enzyme reduction by dihydropyridine nucleotide. A minimal kinetic mechanism has been deduced on the basis of initial velocity measurements and studies on deuterium and tritium isotope effects. Following this scheme, m-hydroxybenzoate and NADH bind to the hydroxylase in a random sequence. The flavohydroxylase is reduced by NADH, and NAD+ is released. Oxygen subsequently binds to and reacts with the reduced flavohydroxylase-m-hydroxybenzoate complex. Following the formation and release of water and gentisate, the oxidized holoenzyme is regenerated. The enzyme has a small (approximately 2-fold) preference for the release of NADH over m-hydroxybenzoate from the enzyme-substrates ternary complex. 相似文献
6.
Abstract
Nitrate reductase was purified from and characterized in a bloom-forming unicellular calcifying alga, Emiliania huxleyi (Haptophyceae). The molecular masses of the native form and the subunit were 514 and 85 kDa, respectively, showing that the
enzyme is a hexamer composed of 6 homologous subunits. The K
m values for NADH and NO3− were 40 μM and 104 μM, respectively. Activity of the reduction of nitrate was very high with reduced
methylviologen and NADH, but no activity was observed with NADPH or reduced flavin mononucleotide; oxidation of NADH was very
high with cytochrome c but did not occur with ferricyanide. These results indicate that Emiliania nitrate reductase is NADH-specific (EC 1.6.6.1), and that among algae and plants its subunit structure and kinetic properties
are unique. 相似文献
7.
Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q0, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K
m-values for both pyridine nucleotides were in the molar range (K
m[NADH]=9.8 M, K
m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP+ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP+ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme
Q0-2,3-dimethoxy-5-methyl-1,4-benzoquinone
- FNR
ferredoxin: NADP+ reductase
- MD
menadione
- MV
methylviologen
- NDH
NAD(P)H dehydrogenase
- PQ
plastoquinone
- PQ10
decylplastoquinone
- SDH
succinate dehydrogenase
- UQ10
decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) 相似文献
8.
The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15+/-6 nmol min-1 mg protein-1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 microM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8-17 nmol min-1 mg protein-1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface. 相似文献
9.
1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (H2NADH) has been investigated as a reduced coenzyme analog in the reaction between trans-4-N,N-dimethylaminocinnamaldehyde (I) (lambdamax 398 nm, epsilonmax 3.15 X 10-4 M-minus 1 cm-minus 1) and the horse liver alcohol dehydrogenase-NADH complex. These equilibrium binding and temperature-jump kinetic studies establish the following. (i) Substitution of H2NADH for NADH limits reaction to the reversible formation of a new chromophoric species, lambdamax 468 nm, epsilonmax 5.8 x 10-4 M-minus 1 cm-minus 1. This chromophore is demonstrated to be structurally analogous to the transient intermediate formed during the reaction of I with the enzyme-NADH complex [Dunn, M. F., and Hutchison, J. S. (1973), Biochemistry 12, 4882]. (ii) The process of intermediate formation with the enzyme-NADH complex is independent of pH over the range 6.13-10.54. Although studies were limited to the pH range 5.98-8.72, a similar pH independence appears to hold for the H2NADH system. (iii) Within the ternary complex, I is bound within van der Waal's contact distance of the coenzyme nicotinamide ring. (iv) Formation of the transient intermediate does not involve covalent modification of coenzyme. Based on these findings, we conclude that zinc ion has a Lewis acid function in facilitating the chemical activation of the aldehyde carbonyl for reduction, and that reduced coenzyme plays a noncovalent effector role in this substrate activating step. 相似文献
10.
Activation of the nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: kinetic characterization and reductant requirement 总被引:4,自引:0,他引:4
The requirements for and kinetics of the activation of the nickel-deficient (apo) CO dehydrogenase from Rhodospirillum rubrum by exogenous nickel have been investigated. The activation is strictly dependent upon the presence of a low-potential one-electron reductant. Sodium dithionite and reduced methylviologen (E degrees' = -440 mV) are suitable reductants, whereas reduced indigo carmine (E degrees' = -125 mV) and the two-electron reductants sodium borohydride, NADH, and dithiothreitol are ineffective in stimulating activation. The midpoint potential for activation was observed at approximately -475 mV. The ability of a reductant to stimulate activation is correlated with the reduced state of the enzyme Fe4-S4 centers. The activation follows apparent first-order kinetics in a saturable fashion, yielding a rate constant of 0.157 min-1 at saturating concentration of nickel. The initial rate at which the enzyme is activated by NiCl2 is also a saturable process, yielding a dissociation constant (KD) of 755 microM for the initial association of nickel and enzyme. Cadmium(II), zinc(II), cobalt(II), and iron(II) are competitive inhibitors of nickel activation with inhibition constants of 2.44, 4.16, 175, and 349 microM, respectively. Manganese(II), calcium(II), and magnesium(II) exhibit no inhibition of activation. 相似文献
11.
Hydrogen transfer between ethanol molecules during oxidoreduction in vivo. 总被引:2,自引:2,他引:0
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T Cronholm 《The Biochemical journal》1985,229(2):315-322
Rates of exchange catalysed by alcohol dehydrogenase were determined in vivo in order to find rate-limiting steps in ethanol metabolism. Mixtures of [1,1-2H2]- and [2,2,2-2H3]ethanol were injected in rats with bile fistulas. The concentrations in bile of ethanols having different numbers of 2H atoms were determined by g.l.c.-m.s. after the addition of [2H6]ethanol as internal standard and formation of the 3,5-dinitrobenzoates. Extensive formation of [2H4]ethanol indicated that acetaldehyde formed from [2,2,2-2H3]ethanol was reduced to ethanol and that NADH used in this reduction was partly derived from oxidation of [1,1-2H2]ethanol. The rate of acetaldehyde reduction, the degree of labelling of bound NADH and the isotope effect on ethanol oxidation were calculated by fitting models to the found concentrations of ethanols labelled with 1-42H atoms. Control experiments with only [2,2,2-2H3]ethanol showed that there was no loss of the C-2 hydrogens by exchange. The isotope effect on ethanol oxidation appeared to be about 3. Experiments with (1S)-[1-2H]- and [2,2,2-2H3]ethanol indicated that the isotope effect on acetaldehyde oxidation was much smaller. The results indicated that both the rate of reduction of acetaldehyde and the rate of association of NADH with alcohol dehydrogenase were nearly as high as or higher than the net ethanol oxidation. Thus, the rate of ethanol oxidation in vivo is determined by the rates of acetaldehyde oxidation, the rate of dissociation of NADH from alcohol dehydrogenase, and by the rate of reoxidation of cytosolic NADH. In cyanamide-treated rats, the elimination of ethanol was slow but the rates in the oxidoreduction were high, indicating more complete rate-limitation by the oxidation of acetaldehyde. 相似文献
12.
Greg B G Moorhead Sarah E M Meek Pauline Douglas Dave Bridges Catherine S Smith Nick Morrice Carol MacKintosh 《European journal of biochemistry》2003,270(6):1356-1362
An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro. 相似文献
13.
The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase. 相似文献
14.
M. Schulz M. Bayer H. White H. Gü nther H. Simon 《Biocatalysis and Biotransformation》1994,10(1):25-36
Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8-9 U mg-1 protein (µmoles mg-1 min-1) for NADPH and 3-4 U mg-1 protein for NADH formation with reduced methylviologen (MV++) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg-1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.
The Km-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.
Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg-1 protein of an NADP+-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV++ regeneration. The regeneration of MV++ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg-1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)+ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.
Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (Ds-(+)-isocitrate), Ls-(-)-isocitrate and 6-phosphogluconate. 相似文献
The Km-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.
Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg-1 protein of an NADP+-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV++ regeneration. The regeneration of MV++ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg-1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)+ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.
Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (Ds-(+)-isocitrate), Ls-(-)-isocitrate and 6-phosphogluconate. 相似文献
15.
Ahlert Schmidt 《Archives of microbiology》1973,93(1):29-52
Summary During sulfate reduction in a cell-free system ofChlorella activated sulfate of APS is transferred to a thiosulfonate reductase. The sulfate thus bound to the thiosulfonate reductase (i.e. bound sulfite) is reduced to bound sulfide in a ferredoxin dependent reaction. This bound sulfide can be used with O-acetylserine as acceptor for cysteine biosynthesis; serine and O-phosphoserine are not used. An assaysystem for thiosulfonate reductase activity using methylviologen dependent reduction of S2O4
2– to S2– is developed and a procedure for isolating thiosulfonate reductase fromChlorella cells is presented. During isolation of thiosulfonate reductase a low weight molecular factor, needed for optimal enzyme activity was lost. The bound sulfite seems to be attached to this factor. Reduction of APS or GS-SO3H to the level of S2– is inhibited by cysteine. 50% inhibition of GS-SO3H reduction was found at a molar cysteine concentration of 6.8×10–5.Abbreviations APS
adenosine-phosphosulfate
- PAPS
3-phosphoadenosine-5-phosphosulfate
- GSH
reduced glutathion
- GS-SO3H
S-sulfoglutathion
- fd
ferredoxin
- Mv
methylviologen
- DTT
dithiothreitol 相似文献
16.
The apoproteins of the streptococcal NADH peroxidase (H2O2----2H2O) and NADH oxidase (O2----2H2O) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6-hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N(1)-protonated p-quinonoid species of 8-mercapto-FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH2 spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EH2.NADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4-thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Furthermore, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH2.NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme. 相似文献
17.
Studies on the mechanism and regulation of C-4 demethylation in cholesterol biosynthesis. The role of adenosine 3′:5′-cyclic monophosphate 总被引:1,自引:1,他引:0
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1. An assay for demethylation has been developed based on the release of tritium from 4,4-dimethyl[3alpha-(3)H]cholest-7-en-3beta-ol (II). 2. The maximum release of (3)H from 3alpha-(3)H-labelled compound (II) in a rat liver microsomal preparation occurs in the presence of NADPH and NAD(+) under aerobic conditions. 3. Incubation of 3alpha-(3)H-labelled compound (II) with NADPH under aerobic conditions leads to the formation of a 3alpha-(3)H-labelled C-4 carboxylic acid. This compound undergoes dehydrogenation on subsequent anaerobic incubation with NAD(+). 4. The (3)H released from the steroid was located in [4-(3)H]nicotinamide and the medium. Incubation with synthetic [4-(3)H(2)]NADH gave a similar result. 5. In the presence of glutamate dehydrogenase and alpha-oxoglutarate part of the (3)H released from the steroid was transferred to glutamate. 6. A series of 3-oxo steroids were reduced equally well by [4-(3)H(2)]NADH and [4-(3)H(2)]NADPH. The reduction of 5alpha-cholest-7-en-3-one was shown to use the 4B H atom from the nucleotide. 7. 3':5'-Cyclic AMP was shown to be a competitive inhibitor of the 3beta-hydroxy dehydrogenase enzyme in the demethylation reaction. 相似文献
18.
The pre-steady-state kinetics of reoxidation of NADH:Q oxidoreductase present in submitochondrial particles has been studied by the freeze-quench method. It was found that at pH 8 only 50% of the Fe-S clusters 2 and 4 and 75% of the clusters 3 were rapidly reoxidised after transient and complete reduction by a pulse of NADH in the presence of excess NADPH. Thus, NADPH keeps 50% of the clusters 2 and 4 and 25% of the clusters 3 permanently reduced at this pH. Since NADH oxidation is nearly optimal at this pH, whereas NADPH oxidation is virtually absent, it was concluded that these permanently reduced clusters were not involved in the NADH oxidation activity. Incomplete reoxidation of the clusters 2, 3 and 4 after a pulse of NADH was also found in the absence of NADPH, both at pH 6.5 and at pH 8. A pulse of NADPH given at pH 6.5, where NADPH oxidation by oxygen is nearly optimal, caused a slow reduction of 50% of clusters 2 and 4 and 30% of the clusters 3, which persisted for a period of at least 15 s. It was concluded that these clusters were not involved in the oxidation of NADPH by oxygen, as catalysed by the particles. As a working hypothesis a dimeric model for NAD(P)H:Q oxidoreductase is proposed, consisting of two different protomers. One of the protomers, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, only reacts with NADH, and its oxidation by ubiquinone is rapid at pH but slow at pH 6.5. The other protomer, containing FMN and the clusters 2, 3 and 4, reacts with both NADH and NADPH and has a pH optimum at 6-6.5 for the reaction with ubiquinone. 相似文献
19.
(21R)-[21-3H]cortisol and (21S)-[21-3H]cortisol were synthesized by reduction of 21-dehydrocortisol by NADH in the presence of 21-hydroxysteroid dehydrogenase. The stereochemistry at carbon 21 was established after cleaving the side chain and oxidizing the resulting two epimers of tritiated glycolate with glycolate oxidase of known (2-pro-S) stereospecificity. From the distribution of radioactivity in the water and glyoxylate produced in this reaction, it was concluded that the reaction of 21-dehydrocortisol with (4S)-[4-3H]NADH catalyzed by 21-hydroxysteroid dehydrogenase results in a transfer of tritium from the 4S position of the nucleotide to form (21S)-[21-3H]cortisol, and that (21R)-[21-3H]cortisol resulted from the enzyme-catalyzed reduction of 21-dehydro[21-3H]cortisol with NADH. Nuclear magnetic resonance studies on both epimers at position 21 of [21-2H]cortisol and of [21-2H]cortisone prepared enzymically identify the transferring 21-pro-S hydrogen as the relatively downfield of the two 21-hydrogen atoms. 相似文献
20.
The stereospecificity of hydrogen transfer in the synthesis of saccharopine from alpha-ketoglutarate and L-lysine catalyzed by saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine: NAD oxidoreductase (L-lysine-forming), EC 1.5.1.7) was examined by using [4A-3H]- and [4B-3H]NADH. The enzyme showed the A-stereospecificity. The NMR analysis of the saccharopine prepared with [4"A-2H]NADH revealed that the label was incorporated into the C-2 of the glutaryl moiety. 相似文献