Cofactor binding to Escherichia coli D-3-phosphoglycerate dehydrogenase induces multiple conformations which alter effector binding

The inhibition of Escherichia coli d-3-phosphoglycerate dehydrogenase by l-serine is positively cooperative with a Hill coefficient of approximately 2, whereas the binding of the inhibitor, l-serine, to the apoenzyme displays positive cooperativity in the binding of the first two serine molecules and negative cooperativity in the binding of the last two serine molecules. An earlier report demonstrated that the presence of phosphate appeared to lessen the degree of both the positive and negative cooperativity, but the cause of this effect was unknown. This study demonstrates that the presence of intrinsically bound NADH was responsible to a substantial degree for this effect. In addition, this study also provides evidence for negative cooperativity in NADH binding and for at least two NADH-induced conformational forms of the enzyme that bind the inhibitor in the physiological range. Successive binding of NADH to the enzyme resulted in an increase in the affinity for the first inhibitor ligand bound and a lessening of both the positive and negative cooperativity of inhibitor binding as compared with that seen in the absence of NADH. This effect was specific for NADH and was not observed in the presence of NAD+ or the substrate alpha-ketoglutarate. Conversely, the binding of l-serine did not have a significant effect on the stoichiometry of NADH binding, consistent with it being a V-type allosteric system. Thus, cofactor-related conditions were found in equilibrium binding experiments that significantly altered the cooperativity of inhibitor binding. Since the result of inhibitor binding is a reduction in the catalytic activity, the binding of inhibitor to these NADH-induced conformers must also induce additional conformations that lead to differential inhibition of catalytic activity.     

Activation of nitrite reductase from Escherichia coli K12 by oxidized nicotinamide-adenine dinucleotide.

Nitrite reductase from Escherichia coli K12 requires the presence of NAD+, one of the products of the reduction of NO2-by NADH, for full activity. The effect is observed with both crude extracts and purified enzyme. NAD+ also acts as a product inhibitor at high concentrations, and plots of initial rate against NAD+ concentration are bell-shaped. The maximum occurs at about 1 mM-NAD+, but increases with increasing NADH concentration. In the presence of 1 mM-NAD+ and saturating NO2-(2mM) the Michaelis constant for NADH is about 16 micron. The Michaelis constant for NO2-is about 5 micron and is largely independent of the NAD+ concentration. Similar but more pronounced effects of NAD+ are observed with hydroxylamine as electron acceptor instead of NO2-. The maximum rate of NADH oxidation by hydroxylamine is about 5.4 times greater than the maximum rate of NADH oxidation by NO2- when assayed with the same volume of the same preparation of purified enzyme. The Michaelis constant for hydroxylamine is 5.3 mM, however, about 1000 times higher than for NO2-. These results are consistent with a mechanism in which the same enzyme-hydroxylamine complex occurs as an intermediate in both reactions.     

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.     

The role of nicotinamide adenine dinucleotide in the inhibition of bovine liver S-adenosylhomocysteine hydrolase by neplanocin A

Neoplanocin A, a cyclopentenyl analog of adenosine, has been shown recently to be a tight binding inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1), exhibiting a stoichiometry of one molecule of inhibitor per molecule of the enzyme tetramer (Borchardt, R. T., Keller, B. T., and Patel-Thombre, U. (1984) J. Biol. Chem. 259, 4353-4358). In the present study a detailed analysis was performed of the possible role of the enzyme-bound NAD+ in the inactivation of AdoHcy hydrolase by neplanocin A. The NAD+/NADH content was quantitated using a fluorescence technique. The native enzyme showed intrinsic fluorescence with an emission maximum at 460 nm when excited at 340 nm, partially due to NADH bound to the enzyme. It was found that the content of NAD+ and NADH in freshly prepared, native enzyme is equal, having a stoichiometry of two nucleotides per enzyme molecule (tetramer). In addition, it was observed that the enzymatic activity of the native enzyme can be increased by about 30% following preincubation with NAD+. Furthermore, it was demonstrated that the mechanism of inhibition of AdoHcy hydrolase by neplanocin A involves the reduction of enzymatically bound NAD+ to NADH. Catalytic activity of the inactivated enzyme could be fully recovered in a time-dependent manner by further incubation with NAD+ (but not NADH). It was also found that inhibition by neplanocin A does not involve dissociation of the bound NAD+ or NADH from the enzyme, but simply reduction of the NAD+ to NADH.     

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.     

Conformational changes and the regulation of glutamate-dehydrogenase activity

1. The effect of NADH and the non-competitive inhibitor GTP on the optical-rotatory-dispersion properties of glutamate dehydrogenase has been studied. 2. Analysis of the data in terms of the a(0) and b(0) parameters of the Moffitt-Yang equation indicates that a conformational change is induced either by NADH or by GTP in the presence of small amounts of NADH. 3. Sedimentation measurements under comparable conditions showed that the enzyme reversibly dissociates into sub-units but that this dissociation is only secondary to the conformational changes. 4. Fluorescence measurements showed that the binding constant of NADH and the number of binding sites on the enzyme increased in the presence of GTP. 5. This is confirmed by studies of fluorescence polarization, which in addition showed that the movement of NADH on the enzyme surface is more restricted in the presence of GTP. 6. The relation of these results to possible regulatory mechanisms is discussed.     

Structure of lactate dehydrogenase inhibitor generated from coenzyme.

Two inhibitors of lactate dehydrogenase generated during NADH storage have been isolated by chromatography. One is a dimer of the dinucleotide where the AMP moiety is unmodified. The other is also generated from NAD+ in the presence of a high concentration of phosphate ions at alkaline pH. This inhibitor was proved to be the addition compound of one phosphate group to position C-4 of the nicotinamide ring of NAD+ by NMR spectroscopy, enzymatic cleavage, and dissociation to NAD+ at neutral pH. This compound is a competitive inhibitor with respect to NAD+ in the presence of the lactate dehydrogenase with a Ki of 2 X 10(-7) M. The interaction of this inhibitor with lactate dehydrogenase is discussed relative to the structure of this enzyme.     

Site-directed mutagenesis identifies catalytic residues in the active site of Escherichia coli phosphofructokinase.

Six active site mutants of Escherichia coli phosphofructokinase have been constructed and characterized using steady-state kinetics. All but one of the mutants (ES222) have significantly lower maximal activity, implicating these residues in the catalytic process. Replacement of Asp127, the key catalytic residue in the forward reaction with Glu, results in an enzyme with wild-type cooperative and allosteric behavior but severely decreased Fru6P binding. Replacement of the same residue with Tyr abolishes cooperativity while retaining sensitivity to allosteric inhibition and activation. Thus, this mutant has uncoupled homotropic from heterotropic allostery. Mutation of Asp103 to Ala results in an enzyme which retains wild-type Fru6P-binding characteristics with reduced activity. GDP, which allosterically activates the wild-type enzyme, acts as a mixed inhibitor for this mutant. Mutation of Thr125 to Ala and Asp129 to Ser produces mutants with impaired Fru6P binding and decreased cooperativity. In the presence of the activator GDP, both these mutants display apparent negative cooperativity. In addition, ATP binding is now allosterically altered by GDP. These results extend the number of active site residues known to participate in the catalytic process and help to define the mechanisms behind catalysis and homotropic and heterotropic allostery.     

An aspartate transcarbamylase lacking catalytic subunit interactions. Study of conformational changes by ultraviolet absorbance and circular dichroism spectroscopy.

A modified form of aspartate transcarbamylase is synthesized by Escherichia coli in the presence of 2-thiouracil which does not exhibit homotropic cooperative interactions between active sites yet retains heterotropic cooperative interactions due to nucleotide binding. The conformational changes induced in the modified enzyme by the binding of different ligands (substrates, substrate analogs, a transition state analog, and nucleotide effectors) were studied using ultraviolet absorbance and circular dichroism difference spectroscopy. Comparison of the results for the modified enzyme and its isolated subunits to those for the native enzyme and its isolated subunits showed that the conformational changes detected by these methods are qualitatively similar in the two enzymes. Comparison of the absorbance difference spectra due to the binding of a transition substrate analog to the intact native or modified enzymes to the corresponding results for the isolated subunits suggested that ligand binding causes an increased exposure to solvent of certain tyrosyl and phenylalanyl residues in the intact enzymes but not in the isolated subunits. This result is consistent with a diminution of subunit contacts due to substrate binding in the course of homotropic interactions in the native enzyme. Such conformational changes, though perhaps necessary for homotropic cooperativity, are not sufficient to cause homotropic cooperativity since the modified enzyme gave identical perturbations. Interactions of the transition state analog, N-(phosphonacetyl)-L-aspartate, with the modified enzyme were studied. Enzyme kinetic data obtained at low aspartate concentrations showed that this transition state analog does not stimulate activity, but rather exhibits the inhibition predicted for the total absence of homotropic cooperative interactions in the modified enzyme. Spectrophotometric titrations of the number of catalytic sites with the transition state analog showed that the modified enzyme and its isolated subunits possess, respectively, four and two high affinity sites for the inhibitor instead of six and three observed in the case of the normal enzyme and its isolated catalytic subunits. These results are correlated with the lower specific enzymatic activities of the modified enzyme and its catalytic subunits compared to the normal corresponding enzymatic species.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sulfhydryl content of L-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation

When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-threonine + NAD(+)----2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12. Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate. The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds. While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate. The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+. NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus L-threonine. Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs. Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+.     

The interaction of liver alcohol dehydrogenase with phenyl adenine dinucleotide, a novel analog of pyridine nucleotide coenzymes.

We have synthesized phenyl adenine dinucleotide (P1-adenosine-5')-P2-(beta-D-ribofuranosylbenzene-5')-pyrophosphate (PhAD), a novel analog of pyridine nucleotide coenzymes. This compound contains a planar aromatic ring, as does NAD+, but lacks a positive charge. PhAD is an inhibitor of horse liver alcohol dehydrogenase, competitive with NADH. PhAD is very similar to NAD+ sterically since both compounds have a planar aromatic ring. However, PhAD resembles NADH in binding to the enzyme because the dissociation constants of both compounds show a parallel increase as the pH is raised, whereas those of NAD+ behave in the opposite manner. These observations indicate that the enzyme differentiates between NAD+ and NADH on the basis of the positive charge on the molecule and not the stereochemical orientation of the reduced nicotinamide ring.     

Pyridine nucleotide interaction with rat liver dihydropteridine reductase.

The interactions of a homogeneous preparation of rat liver dihydropteridine reductase with NADH, NADPH, NAD+, NADP+, and the 1-N6-ethenoadenine derivative of NAD+ have been investigated by fluorescence titration, circular dichroism, equilibrium dialysis, Sephadex G-25 chromatography, and polyacrylamide gel electrophoresis. The procedures indicate that the dimeric enzyme has a definite preference for NADH, but binds only 1 mol of this nucleotide per mol of enzyme. The binary complex of enzyme with NADH is only partially stable to exhaustive dialysis and gel electrophoresis, where it shows greater mobility (0.26) than the free enzyme (0.21); however, the complex can be isolated by Sephadex G-25 chromatography, and characterized with respect to its absorbance spectrum. No ternary complexes are observed when samples of reductase, preincubated with excess NADH, and either the reaction product, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, or the inhibitor, methotrexate, are subjected to polyacrylamide gel electrophoresis.     

The binding of NADH to cytoplasmic aldehyde dehydrogenase after modification with p-nitrophenyl dimethylcarbamate.

Cytoplasmic aldehyde dehydrogenase may be modified by reaction with p-nitrophenyl dimethylcarbamate to give a stable E-X-CO-NMe2 species that is an analogue of the usual labile acyl-enzyme involved in the enzyme's reactions. [X is derived from an enzymic nucleophilic group.] This species still contains the tightly bound NADH that is present in the native enzyme. When further NADH binds to E-X-CO-NMe2 its fluorescence is enhanced over 4 times more than when it binds to unmodified enzyme; this fluorescence is completely unaffected by high propionaldehyde concentration and only slightly affected by p-nitrobenzaldehyde. The modified species has 1.0 NADH binding site in the absence of Mg2+ and 1.67 sites in its presence. The rate of dissociation of E-X-CO-NMe2.NADH is biphasic (k 3.4 and 1.8 min-1) and is considerably lower than that of E.NADH; the presence of Mg2+ slows the process even more (k 0.47 and 0.37 min-1). The implications of these studies towards a greater understanding of the nature of aldehyde dehydrogenase-catalysed reactions are discussed.     

Catalytic properties of streptococcal NADH oxidase containing artificial flavins.

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.     

Selective release and function of one of the two FMN groups in the cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

The soluble, cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H2-NAD+ reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN-b) firmly bound, but it lost its physiological activity--the reduction of NAD+ by H2. This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H2 was not dependent on the presence of FMN-a. Enzyme devoid of FMN-a could not be activated by NADH. As NADH-dehydrogenase activity was not dependent on the presence of FMN-a, and because FMN-b did not dissociate from the reduced enzyme, we conclude that FMN-b is functional in the NADH-dehydrogenase activity catalyzed by the HoxFU dimer. The possible function of FMN-a as a hydride acceptor in the hydrogenase reaction catalyzed by the HoxHY dimer is discussed.     

The interactions of adenylates with allosteric citrate synthase.

Evidence is presented that a number of derivatives of adenylic acid may bind to the allosteric NADH binding site of Escherichia coli citrate synthase. This evidence includes the facts that all the adenylates inhibit NADH binding in a competitive manner and that those which have been tested protect an enzyme sulfhydryl group from reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) in the same way that NADH does. However, whereas NADH is a potent inhibitor of citrate synthase, most of the adenylates are activators. The best activator, ADP-ribose, increases the affinity of the enzyme for the substrate, acetyl-CoA, and saturates the enzyme in a sigmoid manner. A fluorescence technique, involving the displacement of 8-anilino-1-naphthalenesulfonate from its complex with citrate synthase, is used to obtain saturation curves for several nucleotides under nonassay conditions. It is found that acetyl-coenzyme A, coenzyme A, and ADP-ribose all bind to the enzyme cooperatively, and that the binding of each becomes tighter in the presence of KCl, the activator, and oxaloacetic acid (OAA), the second substrate. Another inhibitor, alpha-ketoglutarate, can complete with OAA in the absence of KCl but not in its presence. The nature of the allosteric site of citrate synthase, and the modes of action of several activators and inhibitors, are discussed in the light of this evidence.     

The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12.

Citrate synthase from Escherichia coli enhances the fluorescence of its allosteric inhibitor, NADH, and shifts the peak of emission of the coenzyme from 457 to 428 nm. These effects have been used to measure the binding of NADH to this enzyme under various conditions. The dissociation constant for the NADH-citrate synthase complex is about 0.28 muM at pH 6.2, but increases toward alkaline pH as if binding depends on protonation of a group with a pKa of about 7.05. Over the pH range 6.2-8.7, the number of binding sites decreases from about 0.65 to about 0.25 per citrate synthase subunit. The midpoint of this transition is at about pH 7.7, and it may be one reflection of the partial depolymerization of the enzyme which is known to occur in this pH range. A gel filtration method has been used to verify that the fluorescence enhancement technique accurately reveals all of the NADH molecules bound to the enzyme in the concentration range of interest. NAD+ and NADP+ were weak competitive inhibitors of NADH binding at pH 7.8 (Ki values greater than 1 mM), but stronger inhibition was shown by 5'-AMP and 3'-AMP, with Ki values of 83 +/- 5 and 65 +/- 4 muM, respectively. Acetyl-CoA, one of the substrates, and KCl, an activator, also inhibit the binding in a weakly cooperative manner. All of these effects are consistent with kinetic observations on this system. We interpret our results in terms of two types of binding site for nucleotides on citrate synthase: an active site which binds acetyl-CoA, the substrate, or its analogue 3'-AMP; and an allosteric site which binds NADH or its analogue 5'-AMP and has a lesser affinity for other nicotinamide adenine dinucloetides. When the active site is occupied, we propose that NADH cannot bind to the allosteric site, but 5'-AMP can; conversely, when NADH is the in the allosteric site, the active site cannot be occupied. In addition to these two classes of sites, there must be points for interaction with KCl and other salts. Oxaloacetate, the second substrate, and alpha-ketoglutarate, an inhibitor whose mode of action is believed to be allosteric, have no effect on NADH binding to citrate synthase at pH 7.8. When NADH is bound to citrate synthase, it quenches the intrinsic tryptophan fluorescence of the enzyme. The amount of quenching is proportional to the amount of NADH bound, at least up to a binding ratio of 0.50 NADH per enzyme subunit. This amount of binding leads to the quenching of 53 +/- 5% of the enzyme fluorescence, which means that one NADH molecule can quench all the intrinsic fluorescence of the subunit to which it binds.     

Salts- induced oxidase activity of lipoamide dehydrogenase from pig heart.

A weak NADH oxidase activity of lipoamide dehydrogenase at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3 to 5.5 without changing the maximum activity. The optimum pH is similarly shifted to 5.6 when sulfhyldryl groups of the enzyme are oxidized in the presence of small amount of cupric ion. The NADH: lipoamide and NADH: p-benzoquinone reductase activities are strongly inhibited by KI but both are increased by the presence of (NH4)2SO4. The known intermediate having a charge-transfer band at 530 nm can be seen upon an addition of NADH to the enzyme in the presence of (NH4)2SO4 but not in the presence of KI. The enzyme flavin is reductase by a stoichiometric amount of NADH when KI is present.     

Carvedilol inhibits the exogenous NADH dehydrogenase in rat heart mitochondria

There are several reports on the oxidation of external NADH by an exogenous NADH dehydrogenase in the outer leaflet of the inner membrane of rat heart mitochondria. Until now, however, little was known about its physiological role in cellular metabolism. The present work shows that carvedilol (?1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl)amino]-pro - panol-(2)?) is a specific inhibitor of an exogenous NADH dehydrogenase in rat heart mitochondria. Carvedilol does not affect oxygen consumption linked to the oxidation of succinate and internal NADH. It is also demonstrated that the inhibition of exogenous NADH dehydrogenase by carvedilol is accompanied by the inhibition of alkalinization of the external medium. In contrast to the addition of glutamate/malate or succinate, exogenous NADH does not generate a membrane potential in rat heart mitochondria, as observed with a TPP(+) electrode. It is also demonstrated that the oxygen consumption linked to NADH oxidation is not due to permeabilized mitochondria, but to actual oxidase activity in the inner membrane. The enzyme has a K(m) for NADH of 13 microM. Carvedilol is a noncompetitive inhibitor of this external NADH dehydrogenase with a K(i) of 15 microM. Carvedilol is the first inhibitor described to this organospecific enzyme. Since this enzyme was demonstrated to play a key role in the cardiotoxicity of anticancer drugs of the anthracycline family (e.g., adriamycin), we may suggest that the administration of carvedilol to tumor patients treated with adriamycin might be of great help in the prevention of the cardioselective toxicity of this antibiotic.