The conformation of adenosine diphosphoribose and 8-bromoadenosine diphosphoribose when bound to liver alcohol dehydrogenase.

8-Bromo-adenosine diphosphoribose (br8 ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr8AD+) which are analogues of the coenzyme NAD+, were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in hydrogen transport and br8ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase. X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution. The conformations of these analogues were determined from the X-ray data. It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD+, when NAD+ is bound to lactate and malate dehydrogenase. br8ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD+, in contrast to the syn conformation found in 8-bromo-adenosine. The overcrowding at the 8-position is relieved in br8ADP-Rib by having the ribose in the 2' endo condormation instead of the usual 3' endo as in ADP-Rib and NAD+.     

Alkylation of 6-phosphogluconate dehydrogenase from Candida utilis with coenzyme analogues.

The mechanism of the inactivation of 6-phosphogluconate dehydrogenase from Candida utilis with two coenzyme analogues can be differentiated on the basis of kinetic studies and of the properties of the inactivated enzyme. 3-Chloroacetylpyridine--adenine dinucleotide phosphate is clearly an affinity label and 3-choloroacetylpyridine--adenine dinucleotide a second-order reagent. For 3-chloroacetylpyridine--adenine dinucleotide phosphate, there is a loss of one thiol per subunit at complete inactivation whereas for 3-chloroacetylpyridine--adenine dinucleotide 2.7 thiol groups are lost. The fluorescence of the protein is quenched after alkylation by 3-chloroacetylpyridine--adenine dinucleotide phosphate and there is no quenching after the inactivation with 3-chloroacetylpyridine--adenine dinucleotide.     

Interactions of nicotinamide-adenine dinucleotide phosphate analogues and fragments with pigeon liver malic enzyme. Synergistic effect between the nicotinamide and adenine moieties.

The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5'-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3'-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2'-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2'-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

Depolymerisation of F-actin to G-actin and its repolymerisation in the presence of analogs of adenosine triphosphate.

The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring. The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect. Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide--8-bromoadenine dinucleotide, nicotinamide--8-thioadenine dinucleotide and nicotinamide 1: N6-ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes. Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding. The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary by not ternary complexes. The case of 1,4,5,6-tetrahydronicotinamide--adenine dinucleotide which does not give ternary complexes like NADH is discussed. The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described. The protective effects of coenzyme analogues towards essential -SH group were also studied.     

Induction and Regulation of a Nicotinamide Adenine Dinucleotide-Specific 6-Phosphogluconate Dehydrogenase in Streptococcus faecalis

Streptococcus faecalis grown with glucose as the primary energy source contains a single, nicotinamide adenine dinucleotide phosphate (NADP)-specific 6-phosphogluconate dehydrogenase. Extracts of gluconate-adapted cells, however, exhibited 6-phosphogluconate dehydrogenase activity with either NADP or nicotinamide adenine dinucleotide (NAD). This was shown to be due to the presence of separate enzymes in gluconate-adapted cells. Although both enzymes catalyzed the oxidative decarboxylation of 6-phosphogluconate, they differed from one another with respect to their coenzyme specificity, molecular weight, pH optimum, K(m) values for substrate and coenzyme, and electrophoretic mobility in starch gels. The two enzymes also differed in their response to certain effector ligands. The NADP-linked enzyme was specifically inhibited by fructose-1,6-diphosphate, but was insensitive to adenosine triphosphate (ATP) and certain other nucleotides. The NAD-specific enzyme, in contrast, was insensitive to fructose-1,6-diphosphate, but was inhibited by ATP. The available data suggest that the NAD enzyme is involved primarily in the catabolism of gluconate, whereas the NADP enzyme appears to function in the production of reducing equivalents (NADPH) for use in various reductive biosynthetic reactions.     

Purification and kinetic characterization of 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe.

6-Phosphogluconate dehydrogenase is the pivotal enzyme that links the gluconate route and the oxidative phase of the pentose phosphate pathway in Schizosaccharomyces pombe. The enzyme differs from the known 6-phosphogluconate dehydrogenases of other sources in that the Schizosaccharomyces enzyme is tetrameric having a subunit mass of 38 kDa, that it requires NADP+ obligatorily for activity, and that it can be activated by divalent metal ions such as Co2+ and Mn2+. Steady-state kinetic studies were undertaken. Initial rate and product inhibition results suggest that 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe catalyzes NADP(+)-linked oxidative decarboxylation of 6-phosphogluconate by an equilibrium random mechanism with two independent binding sites, namely one site for the nicotinamide coenzyme, NADP+/NADPH, and another site for 6-phosphogluconate-D-ribulose-5-phosphate and for CO2. Studies of pH dependence implicated a basic residue with a pK value of 7.4 in the binding of 6-phosphogluconate and an acidic residue with a pK value of 6.7 in the cation-mediated interaction of NADP+ with the enzyme.     

Magnetic-resonance studies of the geometry of bound substrate, coenzyme and activator on bovine-liver glutamate dehydrogenase.

ADP and ATP with a spin-label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin-label at a site in the active centre[Andree and Zantema, (1978) Biochemistry, 17, 778--783]. The spin-labelled activators interact with ketone spin-labelled glutamate dehydrogenase in the same way as with native glutamate dehydrogenase relative to the activator site, but show a stronger binding to the coenzyme site. Upon binding to the coenzyme site a spin-spin interaction between the ketone spin-label and the spin-labelled activators is observed. Nuclear magnetic resonance studies of the linewidth of 2-oxoglutarate and NADP+ bound to their functional sites on glutamate dehydrogenase without and with spin-labels result in distances between the ligand nuclei and the spin-labels. The results show that NADP+ binds in an open conformation consistent with the conformation in other dehydrogenases. The activator ADP binds in the neighbourhood of the active centre, but with very little or no overlap with the coenzyme site.     

The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate.

In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.     

Fluorescence studies of coenzyme binding to 6-phosphogluconate dehydrogenase.

The fluorescence quantum yield of NADPH is enhanced in its complex with 6-phospho-gluconate dehydrogenase, and a further enhancement in the presence of excess 6-phospho-gluconate shows that an abortive ternary complex is formed. There is marked energy transfer from aromatic residues in the enzyme to NADPH in the complexes, as indicated by an excitation maximum at 280 nm in the fluorescence excitation spectrum of the complex. The coenzyme fluorescence enhancement has been used to determine the dissociation constant for NADPH in the binary and ternary complexes, and the stoichiometry of the complexes, from the results of fluorescence titrations. A new method of analysis of fluorescence titration data is described. The results show that each subunit of the dimeric enzyme binds NADPH independently and with the same affinity. The dissociation constant for the enzyme-coenzyme complex, in phosphate buffer, pH 7.0, is 5.7 μm; the dissociation constant for NADPH in the ternary complex with 6-phosphogluconate is 7.0 μm.     

Coenzyme binding by triphosphopyridine nucleotide dependent isocitrate dehydrogenase from beef liver. Equilibrium and kinetics studies.

The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP) dependent isocitrate dehydrogenase from beef liver cytoplasm was studied by several equilibrium techniques (ultracentrifugation, molecular sieving, ultrafiltration, fluorescence). Two binding sites (per dimeric enzyme molecule) were found with slightly different dissociation constants (0.5 and 0.12 muM) and fluorescence yields (7.7 and 6.3). A ternary complex was formed between enzyme, isocitrate, and NADPH, in which NADPH dissociation constant was 5 muM. On the contrary, no binding of NADPH to the enzyme took place in the presence of magnesium isocitrate. Dialysis experiments showed the existence of 1 NADP binding site/dimer, with a dissociation constant of 26 muM. When NADPH was present with the enzyme in the proportion of 1 molecule/dimer, the dissociation constant of NADP was decreased fourfold, reaching a value quantitatively comparable to the Michaelis constant. The kinetics of coenzyme binding was followed using the stopped-flow technique with fluorescence detection. NADPH binding to the enzyme occurred through one fast reaction (k1 = 20 muM-1 s-1). Dissociation of NADPH took place upon NADP binding; however, equilibrium as well as kinetic data were incompatible with a simple competition scheme. Dissociation of NADPH from the enzyme upon magnesium isocitrate binding was preceded by the formation of a transitory ternary complex in which the fluorescence of NADPH was only about 30% of that in the enzyme-NADPH complex. Then interaction between the conenzymes and the involvement of ternary complexes in the catalytic mechanism are discussed in relation with what is known about the regulatory role of the coenzyme (Carlier, M. F., and Pantaloni, D. (1976), Biochemistry, 15, 1761-1766).     

Schistosoma mansoni and Schistosoma japonicum: comparison of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in adults.

Glucose-6-phosphate dehydrogenase activity in paired adult Schistosoma mansoni is about twice as great as in paired adult Schistosoma japonicum. 2. 6-phosphogluconate dehydrogenase activity accounts for 25.8% of the measured production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in S. japonicum but only 8.6% of the measured production of NADPH in S. mansoni. 3. These data suggest a species difference in 6-phosphogluconate metabolism.     

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.     

An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase.

1. 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.     

Distances among coenzyme and metal sites of NADP+-dependent isocitrate dehydrogenase using resonance energy transfer

When the substrate isocitrate-Mn2+ is present, the fluorescent nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with pig heart NADP+-specific isocitrate dehydrogenase at the coenzyme binding site on one subunit of the dimeric enzyme [Bailey, J. M., & Colman, R. F. (1985) Biochemistry 24, 5367-5377]. The modified enzyme, which retains partial activity, binds 1 mol of NADPH or 1 mol of the coenzyme analogue, reduced thionicotinamide adenine dinucleotide phosphate (TNADPH), per dimer. TNADPH quenches the fluorescence of enzyme-bound 2-BDB-T epsilon A-2',5'-DP with an efficiency of energy transfer of 9.8%. From this value and the spectral properties of the donor and acceptor chromophores, a distance of 32 A was calculated as the average distance between coenzyme sites on the two subunits. Isocitrate dehydrogenase activity requires a divalent metal ion, such as Mn2+, Co2+, or Ni2+. Co2+ and Ni2+ have absorption spectra that overlap the emission spectra of enzyme-bound 2-BDB-T epsilon A-2',5'-DP. In the presence of isocitrate, each of these two metal ions quenches the fluorescence of the enzyme-bound reagent with an efficiency of energy transfer of 28-29%. From this value and the spectral characteristics of the energy donor and acceptors, an average distance of 8.0 A was estimated between the metal-isocitrate site and the labeled coenzyme site. These distances have provided constraints in formulating a model of the spatial arrangement of active-site ligands on isocitrate dehydrogenase.     

Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver

The steady-state kinetics of the oxidative decarboxylation of 6-phosphogluconate catalysed by 6-phosphogluconate dehydrogenase from sheep liver in triethanolamine and phosphate buffers (pH 7.0) have been reinvestigated. In triethanolamine buffer the enzyme is inhibited by high NADP+ concentrations in the presence of low fixed concentrations of 6-phosphogluconate. Data are consistent with an asymmetric sequential mechanism in which NADP+ and 6-phosphogluconate bind randomly and product release is ordered. The pathway through the enzyme--6-phosphogluconate complex appears to be preferred in triethanolamine buffer. Pre-steady-state studies of the oxidative decarboxylation reaction at pH 6.0-8.0 show that hydride transfer is greater than 900 s-1. After the fast formation of NADPH in amounts equivalent to about half of the enzyme-active-centre concentration, the rate of NADPH formation is equal to the steady-state rate. Two possible interpretations are considered. Rapid fluorescence measurements of the displacement of NADPH from its complex with the enzyme at pH 6.0 and 7.0 indicate that the dissociation of NADPH is fast (greater than 800 s-1) and cannot be the rate-limiting step in oxidative decarboxylation. Coenzyme binding studies at equilibrium have been extended to include the determination of the dissociation constants for the binary complexes of enzyme with NADPH and NADP+ at pH 6.0-8.0 and the dissociation constant for NADPH in the ternary enzyme--6-phosphogluconate--NADPH complex in triethanolamine buffer, pH 7.0.     

Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.     

Human Brain 6-Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.     

6-Phosphogluconate dehydrogenase. Purification and kinetics.

A method is described for the isolation and purification of 6-phosphogluconate dehydrogenase from pig liver. The molecular weight is estimated at 83,000 and that of the subunits is 42,000 as determined by gel electrophoresis. The pH maximum is 8.5 in 50 mM glycine/NaOH buffer and from 7.5 to 10 in 50 mM phosphate buffer at 30 degrees. Magnesium ion is not required for activity and acts as an inhibitor at concentrations above 20 mM. A cellular fractionation study indicates that this enzyme is located almost entirely within the soluble portion of the cytoplasm. Kinetic studies have been done in 50 mM glycine buffer, pH 8.5, at 30 degrees. The data are consistent with a sequential mechanism in which NADP+ is added first, followed by 6-phosphogluconate, and the products are released in the order, CO2, ribulose 5-phosphate, and NADPH. The Michaelis constant is 13.5 muM for 6-phosphogluconate. Dissociation constants are 4.8 muM for NADP+ and 5.1 muM for NADPH.     

Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase is a potential target for new drugs against African trypanosomiasis. Phosphorylated aldonic acids are strong inhibitors of 6-phosphogluconate dehydrogenase, and 4-phospho-d-erythronate (4PE) and 4-phospho-d-erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei enzyme. Binding of the substrate 6-phospho-d-gluconate (6PG), the inhibitors 5-phospho-d-ribonate (5PR) and 4PE, and the coenzymes NADP, NADPH and NADP analogue 3-amino-pyridine adenine dinucleotide phosphate to 6-phospho-d-gluconate dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (K(d) = 5 microm) and its analogues (K(d) =1.3 microm and K(d) = 2.8 microm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the coenzymes is enthalpy driven. Oxidized coenzyme and its analogue, but not reduced coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the coenzymes, whereas binding of 4PE decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar manner, the K(d) value of 4PE decreases by two orders of magnitude in the presence of the coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the enzyme, suggesting that the inhibitor forces the catalytic lysine 185 into the protonated state.