The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme.

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.     

Inhibition and inactivation of bovine mammary and liver UDP-galactose-4-epimerases.

Bovine liver and mammary UDP-galactose-4-epimerases were investigated with respect to various inhibitors and inactivators. Uridine nucleotides and NADH are potent inhibitors with Ki values in the low micromolar range. The NAD+/NADH ratio may be an important physiological control mechanism for it affects markedly the activity of the enzyme with 50% inhibition occurring at a ratio of 20:1. In the presence of uridine nucleotides binding of NADH to the epimerases is enhanced. Consequently, the effect of changes in the NAD+/NADH ratio in vivo would not be immediately apparent as uridine nucleotides would slow down the displacement of NADH by NAD+. Neither uridine nor galactose 1-phosphate inhibits the purified enzymes as previously reported with the impure liver enzyme. Uridine nucleotides provide almost total protection against the apparent first order inactivation of the epimerases by trypsin and allow determination of dissociation constants. NAD+ partially protects against trypsin inactivation. Inactivation with various sulfhydryl reagents is complex and the results indicate that at least three sulfhydryl groups may be modified before total inactivation occurs. Partial inactivation occurs upon modification of the epimerases with 2-hydroxy-5-nitrogenzyl bromide. Some protection against this modification is provided by the combination of NAD+ and UDP.     

\vec H^ + /2\bar e Stoichiometry of the NADH:Ubiquinone Reductase Reaction Catalyzed by Submitochondrial Particles

Mitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton translocation into inside-out submitochondrial particles. Here we describe a method for determining the stoichiometric ratio (n) for the coupled reaction of NADH oxidation by the quinone acceptors. Comparison of the initial rates of NADH oxidation and alkalinization of the surrounding medium after addition of small amounts of NADH to coupled particles in the presence of Q₁ gives the value of n = 4. Thermally induced deactivation of Complex I [1,2] results in complete inhibition of the NADH oxidase reaction but only partial inhibition of the NADH:Q₁-reductase reaction. N-Ethylmaleimide (NEM) prevents reactivation and thus completely blocks the thermally deactivated enzyme. The residual NADH:Q₁-reductase activity of the deactivated, NEM-treated enzyme is shown to be coupled with the transmembraneous proton translocation (n = 4). Thus, thermally induced deactivation of Complex I as well as specific inhibitors of the endogenous ubiquinone reduction (rotenone, piericidin A) do not inhibit the proton translocating activity of the enzyme.     

NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain

Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity. In comparison, membranes from E. coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity. Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol. Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH. Inside-out membrane vesicles from E. coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate. Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity. The data provide a strong indication that the E. coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.     

The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme.

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.     

Nicotinamide-adenine dinucleotide inhibition of pig kidney alkaline phosphatase.

1. The interaction of NAD+, NADH and various nucleotide analogues with pig kidney alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1) has been investigated by kinetic means. Some inhibitors act uncompetitively whereas others markedly increase the slopes of double reciprocal plots suggesting they have some affinity for the free enzyme. 2. The compounds seem to bind to alkaline phosphatase through interactions of their bases with a relatively non-specific region of the enzyme, although it is likely that for those nucleotides having some affinity for the free enzyme there is some attraction between the pyrophosphate backbone and the active site. 3. From studies of the effect of NAD+ and NADH on ATPase activity it was concluded that the substrate inhibition that is characteristic of the ATPase activity of alkaline phosphatase originates from binding of ATP to the site assumed to exist for NAD+ and NADH. The potentiation of NAD+-inhibition of ATPase activity by Mg-2+ is probably a result of the depletion of [ATP-4-] the true substrate. The depletion allows NAD+ to complete more effectively for the active site. 4. Binding of NADH is favoured by protonation of an enzymic group with a pK of approx. 9.0 belonging possibly to a tyrosine residue or a zinc hydrate. 5. A large entropy decrease was found to accompany the binding of NAD+ and NADH to alkaline phosphatase. This may be further evidence of an "induced-fit" mechanism previously suspected because of the synergistic inhibitory effects of adenosine and nicotinamide.     

Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis

The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.     

Carboxymethyl horse-liver alcohol dehydrogenase. Ligand-binding and kinetic properties of the cysteine-46-modified enzyme.

Horse-liver alcohol dehydrogenase was carboxymethylated with iodoacetate, which is known to selectively alkylate cysteine-46 in the polypeptide sequence. Carboxymethyl and native enzyme had the same electrophoretic mobility on starch or polyacrylamide gel, but some separation was achieved when isobutyramide and a low concentration of NADH were present (under these conditions NADH was bound by native enzyme but not by Carboxymethyl enzyme).The Carboxymethyl enzyme formed ternary complexes with NAD⁺ and pyrazole or decanoate. The fluorescence emission of NADH was enhanced 7- to 8-fold (at 410 nm), and a dissociation-constant of 1.7 μM was calculated at pH 7.4; but, in contrast to native enzyme, neither the affinity nor fluorescence were increased by amides (acetamide or isobutyramide).Carboxymethyl alcohol dehydrogenase possesses catalytic activity. Higher alcohols gave maximum velocities up to 7-fold higher than ethanol (reaching nearly 20% of the activity of native enzyme) while [²H]ethanol showed an isotope-rate effect of 3.3. Although the affinity for aldehydes was considerably increased, the maximum velocity of aldehyde-reduction was always at least 20% of that shown by native enzyme, and at pH 9.9 it was almost 2-fold greater than with native enzyme. The rate-limiting step in alcohol-oxidation is likely to be the interconversion of ternary complexes (possibly the hydride-transfer step), while in aldehyde-reduction it could still be the dissociation of the enzyme/NAD⁺ complex. This is also indicated by inhibition experiments with decanoate, pyrazole, and isobutyramide.These results suggest that a major effect of carboxymethylation is upon ternary complexes of enzyme and NADH, which become much more reluctant to form, either by combination of NADH and ligand with the modified enzyme, or by catalytic conversion of the enzyme/NAD ⁺/alcohol complex.     

Purification and properties of crystalline 3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides

1. The purification and crystallization of 3-hydroxybutyrate dehydrogenase from extracts of Rhodopseudomonas spheroides is described. 2. The molecular weight was calculated to be 85000 by sedimentation equilibrium. 3. Although the enzyme is stable at 0-4 degrees , dilute solutions are rapidly inactivated at 37 degrees ; NADH(2) or Ca(2+) ions prevent this inactivation. 4. The enzyme is extremely sensitive to mercurials, but can be protected by NADH(2) or Ca(2+) ions. 5. From studies on p-hydroxymercuribenzoate binding it is estimated that the enzyme contains 5-6 moles of rapidly reacting thiol groups/mole. 6. d-Lactate and dl-2-hydroxybutyrate are competitive inhibitors of d-3-hydroxybutyrate oxidation. 7. The properties of the crystalline enzyme are compared with those of 3-hydroxybutyrate dehydrogenase preparations from other sources.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: activity and role of mitochondria in bundle sheath cells

Mitochondria from bundle sheath cells of the phosphoenolpyruvate carboxykinase-type C4 species Urochloa panicoides were shown to have metabolic properties consistent with a role in C4 photosynthesis predicted from earlier studies. The rate of O2 uptake in response to added malate plus ADP was at least five times the activity observed with NADH, glycine, or succinate. With malate plus ADP the O2 uptake rate averaged about 150 nmol O2 min-1 mg-1 protein, equivalent to about 0.6 mumol min-1 mg-1 of extracted chlorophyll. About half of this activity was apparently phosphorylation-linked with ADP/O2 ratios of about 4. Studies with electron transport inhibitors suggested that about 65% of this malate oxidation is cytochrome oxidase-terminated with a minor component mediated via the alternative oxidase. These mitochondria supported rapid rates of pyruvate production from malate and this activity was also stimulated by ADP but blocked by inhibitors of electron transport. Adding oxaloacetate increased pyruvate production but inhibited O2 uptake. The results were consistent with the notion that in this subgroup of C4 species mitochondrial-located NAD malic enzyme contributes substantially to total C4 acid decarboxylation. This enzyme is apparently also the primary source of NADH necessary to generate the ATP required for phosphoenolpyruvate carboxykinase-mediated oxaloacetate decarboxylation.     

Purification and characterization of thermostable H<Subscript>2</Subscript>O<Subscript>2</Subscript>-forming NADH oxidase from 2-phenylethanol-assimilating <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. KU1309

A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.     

Human glutamic-gamma-semialdehyde dehydrogenase. Kinetic mechanism.

The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.     

Malate dehydrogenase of spinach: Substrate kinetics of different forms

Sephadex G-200 gel filtration of an ammonium sulfate fraction, containing the bulk of NAD-dependent malate dehydrogenase, yields forms of differing MW. Both Mg²⁺ and NADH stabilize the 127000 daltons MW form. K⁺, or incubation with dithioerythritol, cause splitting and partial reaggregation, resulting in MWs ranging between 35000 and 180000 daltons. Chromatography in the presence of dithioerythritol and NADH results in an enzyme with a non-linear reaction rate at low substrate concentrations. Plots of initial velocity vs substrate and cofactor concentration respectively are characterized by two slopes of positive cooperativity separated by an intermediary plateau of negative cooperativity. Gel chromatography in the presence of Mg²⁺ or K⁺ or drastic dilution of the enzyme results in an enzyme with linear reaction rates also at low substrate concentration. Its kinetics are consistent with the view that the enzyme undergoes conformational changes when the substrate concentration is varied.     

Lifetimes and NADH quenching of tryptophan fluorescence in pig heart cytoplasmic malate dehydrogenase.

The time-resolved and steady state fluorescence properties were measured for pig heart cytoplasmic malate dehydrogenase at pH 6.0 and 8.0. The fluorescence decay can be described by two rate processes, according to the functions: I(t) = 0.7e(-t/1.0) + 0.3e(-t/4.4) for the free enzyme and I(t) = 0.7e(-t/0.8) + 0.3e(-t/2.0 for the enzyme . NADH complex. Quenching by NADH of the tryptophan fluorescence is linear. The only effect of pH is to change the association constant for NADH binding; the fluorescence of the free enzyme and the fluorescence quenching by NADH, I-, and acrylamide are unaffected by pH. Thus there are no changes in conformation of the free enzyme or of the NADH complex over the range of pH 6 to 8.     

Purification and characterization of pyruvate dehydrogenase complex from borccoli floral buds.

The pyruvate dehydrogenase complex (PDC) was purified from Brassica oleracea var. italica floral buds to a specific activity of approximately 6 μmol of NADH formed/min/ mg of protein. The PDC had cofactor requirements for NAD⁺, thiamine pyrophosphate, coenzyme A, and a divalent cation (Mg²⁺, Ca²⁺, or Mn²⁺). The enzyme catalyzed the oxidative decarboxylation of pyruvate at a rate threefold faster than 2-oxobutyrate but was inactive toward 2-oxoglutarate. The PDC was competively inhibited by acetyl-CoA against CoA and NADH against NAD⁺. The enzyme was shown to be more sensitive to regulation by NADH than acetyl-CoA.     

Characteristics and actions of NAD(P)H oxidase on the sarcoplasmic reticulum of coronary artery smooth muscle

It has been reported that nonmitochondrial NAD(P)H oxidases make an important contribution to intracellular O2-* in vascular tissues and, thereby, the regulation of vascular function. Topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2-* into the cytosol. It is imperative to clarify the source of intracellular O2-* associated with this enzyme and its physiological significance in vascular cells. The present study hypothesized that an NAD(P)H oxidase on the sarcoplasmic reticulum (SR) in coronary artery smooth muscle (CASM) regulates SR ryanodine receptor (RyR) activity by producing O2-* locally. Western blot analysis was used to detect NAD(P)H oxidase subunits in purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of SR with NADH time dependently produced O2-*, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin and by SOD or its mimetic tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of conversion of NADH to NAD+. In experiments of lipid bilayer channel reconstitution, addition of NADH to the cis solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM, with a maximal increase in channel open probability from 0.0044 +/- 0.0005 to 0.0213 +/- 0.0018; this effect of NADH was markedly blocked in the presence of SOD or tiron or the NAD(P)H oxidase inhibitors diphenylene iodonium, N-vanillylnonanamide, and apocynin. These results suggest that a local NAD(P)H oxidase system on SR from CASM regulates RyR/Ca2+ channel activity and Ca2+ release from SR by producing O2-*.     

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP⁺ or NAD⁺ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP⁺) is better electron donor (or acceptor), while NADH (or NAD⁺) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP⁺, 2′-AMP and and atebrin.     

Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae

A mitochondrial NADH:Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370 mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km = 31 microM and was found to be inhibited by flavone (I50 = 95 microM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s-1). With the natural ubiquinone-6 this value was 500 s-1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0. The amount of enzyme in the cell is subject to glucose repression; it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.     

Studies on the interactions of glycerol dehydrogenase from Bacillus stearothermophilus with Zn2+ ions and NADH

The interactions of the essential divalent cation, Zn2+, with the binary complex formed between glycerol dehydrogenase (glycerol:NAD+ 2-oxidoreductase, EC 1.1.1.6) and its coenzyme NADH have been examined by fluorescence spectroscopy. Both the metallo and non-metallo form of the enzyme bind the coenzyme NADH. The addition of Zn2+ ions to a solution of the binary complex formed between metal-depleted enzyme and NADH results in a rapid increase in fluorescence emission at 430 nm. This has been used to determine the on rate for Zn2+ to the enzyme/binary complex. A dissociation constant of 3.02 +/- 0.25.10(-9) M for the equilibrium between Zn2+ ions and the enzyme has been determined.     

Allosteric Properties of Ox Brain Nicotinamide–Adenine Dinucleotide Dependent Isocitrate Dehydrogenase

Abstract: The kinetic and regulatory properties of a partly purified preparation of ox brain NAD⁺-dependent isocitrate dehydrogenase have been studied at pH 7.5. The enzyme exhibits rate cooperativity with respect to isocitrate but shows normal hyperbolic kinetics with respect to NAD⁺. ADP activates the enzyme by decreasing the substrate concentrations that are necessary to give half-maximal velocity, but it has no effect on the Hill constant for isocitrate unless Mg²⁺ ions are replaced by Mn²⁺ ions in the reaction mixture. Citrate and tricarballylate activate the enzyme in a similar fashion to ADP. Higher concentrations of citrate cause inhibition but this could be overcome by raising the concentration of Mg²⁺ ions, suggesting that the inhibition by this compound might be due to its acting as a chelating agent. NADH and NADPH were competitive inhibitors with respect to NAD⁺ but the product, 2-oxoglutarate, was not inhibitory. γ-Aminobutyrate and a number of other compounds involved in the γ-aminobutyrate pathway had no significant effect on the activity of the enzyme.