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PubMed1991

Apparent ATP-linked succinate thiokinase activity and its relation to nucleoside diphosphate kinase in mitochondrial matrix preparations from rabbit.

The relative abilities of ATP and GTP to support succinyl-CoA synthesis by mitochondrial matrix fractions prepared from rabbit heart and liver mitoplasts were investigated. The activity supported by ATP in rabbit heart preparations was less than 15% of that obtained with GTP, while no ATP-supported activity was observed in rabbit liver preparations. However, the addition of 30 micromolar GDP to matrix fractions from either heart or liver stimulated the ATP-supported activity to 40% of that observed with GTP, and the further addition of bovine liver nucleoside diphosphate kinase in the presence of 8 microM added GDP increased the activity to near that observed with GTP. The specific activity of nucleoside diphosphate kinase assayed directly in mitochondrial matrix from heart was about 15% of the specific activity of ATP-supported succinate thiokinase induced upon adding GDP. Evidence for a complex between nucleoside diphosphate kinase and succinate thiokinase in mitochondrial matrix from rabbit heart was obtained by glycerol density gradient centrifugation. It is proposed that binding of nucleoside diphosphate kinase to succinate thiokinase activates the former enzyme, accounts for the ATP-supported succinyl-CoA synthetase activity observed, and is involved in the channeling of high energy phosphate from GTP produced in the Krebs cycle to the ATP pool.

Adenosine Triphosphate↗
PubMed2005

Biosynthesis of uridine diphosphate N-acetyl-L-fucosamine in a cell-free system from Salmonella arizonae O:59.

The conversion of uridine diphosphate N-acetyl-D-glucosamine into uridine diphosphate N-acetyl-L-fucosamine was demonstrated with enzymes from cytoplasmic fraction of Salmonella arizonae O:59 cells in the presence of NAD+ (NADP+) and NADPH. The reaction product was identified by ion-pair, reverse-phase HPLC with the use of synthetic nucleoside diphosphate sugar standards under conditions specially developed for separation of uridine diphosphate 2-acetamido-2,6-dideoxyhexoses. L-Fucose dehydrogenase from porcine liver was shown to be applicable for determination of N-acetyl-L-fucosamine, this enzyme being used to confirm L-configuration of the amino sugar residue in the sugar nucleotide formed.

Animals↗
PubMed1990

Nucleoside diphosphate kinase from Myxococcus xanthus. II. Biochemical characterization.

The gene that encodes the 16-kDa GTP-binding protein from Myxococcus xanthus has been cloned, and its DNA sequence has been determined. The gene has been expressed in Escherichia coli by using the lacZ promoter, and its gene product was overproduced (Muñoz-Dorado, J., Inouye, M., and Inouye, S. (1990) J. Biol. Chem. 265, 2702-2706). The gene product thus overproduced in E. coli was purified to homogeneity by a simple four-step procedure and crystallized. Gel filtration of the purified protein revealed that the protein forms a complex of an apparent molecular weight of 50,000, indicating that it exists as a trimer in the cell. It was found that the purified protein can bind not only GTP, but also equally well the other nucleoside diphosphates and triphosphates with no specificity for either the base or the sugar. Nucleoside monophosphates, Pi, and pyrophosphate do not bind to the protein. In the presence of Mg2+, the protein hydrolyzes nucleoside triphosphates to diphosphates and Pi. However, in the presence of EDTA, most of the phosphate remains bound to the protein. The phosphorylated protein can then transfer the phosphate group to a nucleoside diphosphate to form the corresponding nucleoside triphosphate in the presence of Mg2+. The reaction is reversible, and it is considered to occur by a two-step ping-pong mechanism. These results unambiguously demonstrate that the M. xanthus 16-kDa GTP-binding protein is a nucleoside diphosphate kinase.

Adenosine Triphosphate↗
PubMed1983

Mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Evidence for 3'-C--H bond cleavage.

Incubation of the pyrimidine [3'-3H]UDP with ribonucleotide reductase resulted in an isotope effect on the conversion to dUDP which varied as a function of pH and allosteric effectors (pH, kH/kT, effector): 6.6, 4.7, ATP; 7.6, 3.3, ATP; 7.6, 2.6, dATP; 7.6, 2.0, TTP; 8.4, 2.8, ATP. During this reaction 3H2O was also released. The lower the pH of the reaction, the larger the isotope effect, and the smaller the amount of 3H2O produced. At 50% conversion of UDP to dUDP and at pH 7.6, approximately 0.5% of total 3H present in solution was volatilized, while at pH 8.4, approximately 0.9% was volatilized. Similar experiments in which the purine [3'-3H]ADP was incubated with ribonucleotide reductase also resulted in an isotope effect on its conversion to dATP which varied as a function of pH (pH, kH/kT with dGTP as an effector); 6.6, 1.9; 7.6, 1.7; 8.6, 1.4. Furthermore, 3H2O was also released as a function of the extent of the reaction. At 50% turnover and pH 7.6, approximately 0.6% of 3H2O was volatilized, while at pH 8.6 approximately 1.25% was released. Two control experiments in which either the B1 subunit of ribonucleotide reductase was inactivated with 2'-chloro-2'-deoxyuridine 5'-diphosphate or the B2 subunit of ribonucleotide reductase was inactivated with 2'-azido-2'-deoxyuridine 5'-diphosphate and then the enzyme incubated with [3'-3H]ADP or [3'-3H]UDP indicated that in neither case was 3H released. Both B1 and B2 subunits are required for cleavage of the 3'-C--H bond. Incubation of [3'-3H]dADP or [3'-3H]dUDP with ribonucleotide reductase produced no measurable release of 3H. These data clearly indicate that conversion of a purine or pyrimidine diphosphate to a deoxynucleotide diphosphate by Escherichia coli ribonucleotide reductase requires cleavage of the 3'-C--H bond of the substrate. The fate of the 3'-H of the substrate was also determined. Incubation of [3'-2H]UDP with ribonucleotide reductase resulted in the production of [3'-2H]dUDP.

Allosteric Regulation↗
PubMed1994

Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions.

nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although the mechanisms remain undefined. All Nm23 proteins contain nucleoside diphosphate kinase (NDPK) activity whose significance to these regulatory effects is not yet evident. The protein product of the human nm23-H2 gene functions in vitro both as a nucleoside diphosphate kinase enzyme (NDPK-B; Gilles, A.-M., Presecan, E., Vonica, A. and Lascu, I. (1991) J. Biol. Chem. 266, 8784-8789) and as a transcription factor (PuF; Postel, E. H., Berberich, S. J., Flint, S. J. and Ferrone, C. A. (1993) Science 261, 478-480). To understand the significance of these two biochemical activities to NM23-H2 function, we have investigated the relationship between the DNA binding and transcriptional activity of NM23-H2 and its NDPK function. Using site-directed mutagenesis of the cDNA encoding NM23-H2, we have created a mutant substituting for the amino acid histidine 118, the presumed site of phosphorylation in the formation of the phosphoenzyme intermediate, the nonphosphorylatable amino acid phenylalanine. The H118F mutant protein is shown to be catalytically inactive as measured both in a radioisotopic assay that detects formation of the phosphorylated enzyme intermediate and in a coupled enzyme assay that indicates nucleoside diphosphate formation. These results confirm that histidine 118 is the critical residue for NDPK-B activity. In addition, the H118F mutant protein lacking enzymatic activity displayed normal DNA binding affinity for the c-myc promoter in electrophoretic mobility shift assays, and retained full transcriptional activity using the c-myc gene in vitro. These results indicate a lack of correlation between nucleoside diphosphate kinase activity of nm23-H2 on the one hand, and its DNA binding and transcriptional activity on the other, suggesting that the nm23-H2 gene encodes a bifunctional protein molecule.

Base Sequence↗
PubMed2002

Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase.

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of terminal phosphates from nucleoside triphosphates to nucleoside diphosphates to yield nucleotide triphosphates. The present study was undertaken to localize and characterize the mitochondrial isoform of NDPK (mNDPK) in the pancreatic beta cell since it could contribute to the generation of mitochondrial nucleotide triphosphates and, thereby, to the mitochondrial high-energy phosphate metabolism of the pancreatic beta cell. Mitochondrial fractions from the insulin-secreting beta cells were isolated by differential centrifugation. mNDPK activity was assayed as the amount of [(3)H]GTPgammaS formed from ATPgammaS and [(3)H]GDP. Incubation of isolated mitochondrial extracts with either [gamma-(32)P]ATP or GTP resulted in the formation [(32)P]NDPK, which could be immunoprecipitated by an anti-NDPK serum. mNDPK exhibited saturation kinetics with respect to its nucleoside diphosphate acceptors and nucleoside triphosphate donors and sensitivity to known inhibitors of NDPK (e.g., uridine diphosphate and cromoglycate). By Western blot analyses, at least three isoforms of NDPK were identified in various subcellular fractions of the beta cell. The nm23-H1 (NDPK-A) was predominantly soluble whereas nm23-H2 (NDPK-B) was associated with the soluble as well as membranous fractions. The mitochondrial isoform of NDPK, nm23-H4, was uniformly distributed in the beta cell mitochondrial subfractions. A significant amount of NDPK (as determined by the catalytic activity and immunological methods) was recovered in the immunoprecipitates of mitochondrial fraction precipitated with an antiserum directed against succinyl-CoA synthetase (SCS), suggesting that NDPK might remain complexed with SCS. We provide the first evidence for the localization of a mitochondrial isoform of the NDPK in the islet beta cell and thus offer a potential mechanism for the generation of intramitochondrial GTP which, unlike ATP, is not transported into mitochondria via the classical nucleotide translocase. Further work will be required to determine the importance of the NDPK/SCS complex to normal beta cell function in the secretion of insulin.

Animals↗
PubMed1997

Cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol import into mitochondria through mitochondrial membrane contact sites in permeabilized rat liver hepatocytes.

The location of cytidine-5'-diphosphate-1,2-diacylsn-glycerol import into mictochondria was examined in permeabilized rat liver hepatocytes. The model utilized the ability of intact mitochondria to freely import [14C]glycerol-3-phosphate. Import of cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol was measured by the synthesis of phosphatidyl[14C]glycerol. Phosphatidyl [14C]glycerol was synthesized in a time- and concentration-dependent manner in the presence of cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol. The presence of ATP in the incubations stimulated phosphatidyl[14C]glycerol formation. The presence of adriamycin, at concentrations that block import of proteins into mitochondria, inhibited the formation of phosphatidyl [14C]glycerol. In addition, adriamycin, inhibited in vitro biosynthesis of phosphatidyl[14C]glycerol in mitochondrial but not microsomal fractions. 1,4-Dinitrophenol, which may decrease the number of mitochondrial inner and outer membrane contact sites, inhibited formation of phosphatidyl[14C]glycerol in permeabilized hepatocytes. The results demonstrate that cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol may be imported into mitochondria through inner and outer mitochondrial membrane contact sites in hepatocytes.

Animals↗
PubMed1985

Platelet sensitivity in vitro to adenosine-5'-diphosphate and prostacyclin and diabetic retinopathy.

Platelet sensitivity to adenosine diphosphate and prostacyclin in diabetes has been assessed using collision theory and the concept of 'sticking probability' (the probability of particle union). Twenty Type 1 (insulin-dependent) diabetic men (10 with no or minimal retinopathy and a matched group of 10 with proliferative retinopathy) and 10 age-matched nondiabetic men were studied. Platelets from the 20 diabetic patients required, on average, 37% less adenosine diphosphate to achieve a sticking probability of 0.5 (ED50) compared with platelets from the non-diabetic subjects (medians 1.50 and 0.95 mumol/l, respectively; p less than 0.01). The platelet prostacyclin response was assessed by the dissociation constant (Ki) for inhibition of adenosine diphosphate-induced aggregation. Platelets from the diabetic patients had similar prostacyclin sensitivity to those from the non-diabetic subjects (medians 0.42 and 0.42 respectively). Diabetic patients with and without retinopathy had similar platelet sensitivity to both adenosine diphosphate and prostacyclin.

Adenosine Diphosphate↗
PubMed1975

Role of cytidine triphosphate and cytidine diphosphate in promoting inositol entry into microsomal phosphatidylinositol.

The Mn2+ activated incorporation of myo-inositol-3H into subfractions of phosphatidylinositol in rat liver microsomes was studied in the presence and absence of cytidine triphosphate or cytidine diphosphate choline using phosphate buffer. The distribution of labeled inositol among molecular species of microsomal phosphatidylinositol was also investigated in vivo. In other experiments, the release of radioactivity from microsomes labeled with inositol-3H in the phospholipid was measured after the addition of Mn2+, unlabeled inositol, and cytidine nucleotide. Similar chase experiments were conducted with microsomes containing phosphatidylcholine-14C or phosphatidylethanolamine-14C. The addition of cytidine triphosphate or cytidine diphosphate choline stimulated the rate of inositol-3H entry into microsomal phosphatidylinositol by 3.5 to 4-fold and the monoenoic plus dienoic, trienoic, tetraenoic, and polyenoic species contained 6-7, 6, 78-81, and 7-9%, of radioactivity, respectively. These latter patterns were very similar to those observed among the corresponding molecular species when the Mn2+ stimulated entry of free inositol into phospholipid was studied in the absence of added cytidine nucleotide. In chase experiments, the release of radioactivity from phospholipid in the presence of cytidine trephosphate or cytidine diphosphate choline was greatly enhanced by the addition of free inositol when microsomes containing phosphatidylinositol-3H, but not phosphatidylcholine-14C or phosphatidylethanolamine-14C, were employed. Therefore, under the present conditions, cytidine triphosphate and cytidine diphosphate choline appear to stimulate the entry of inositol into phosphatidylinositol by enhancing the Mn2+ activated exchange reaction in rat liver microsomes. The results suggest further that phosphatidylinositol is the preferred substrate when this reaction is stimulated by cytidine nucleotide.

Animals↗
PubMed1985

Evidence for a different metabolic behaviour of cytidine diphosphate choline after oral and intravenous administration to rats.

Radioactivity plasma decay was studied in rats after intravenous and oral administration of cytidine diphosphate [methyl-14C]choline at doses of 25 and 300 mg/kg. The kinetics fitted well with a two compartment open model and showed a long lasting elimination phase with a half-life ranging from 2.0 to 2.6 days for the two doses and the two administration routes. Absorption of cytidine diphosphate choline radioactivity was complete after oral treatment with the low dose and accounted for 94.5% of the dose when 300 mg/kg of cytidine diphosphate [methyl-14C]choline were administered. However the distribution of radioactivity in tissues, urine and expired air suggest metabolic differences, at least from a quantitative point of view, between the oral and intravenous treatments. In particular, the higher excretion of radioactivity associated with trimethylamine in urine found when cytidine diphosphate [methyl-14C]choline was given orally, suggest that the compound may be metabolized, at least in part, previous to its gastrointestinal absorption.

Administration, Oral↗
PubMed1995

NMR studies of binding of 5-FdUDP and dCDP to ribonucleoside-diphosphate reductase from Escherichia coli.

5-Fluoro-2'-deoxyuridine-5'-diphosphate (5-FdUDP) has been synthesised using an original route, previously applied to the synthesis of natural nucleoside diphosphates. The interaction between 5-FdUDP and the enzyme ribonucleoside-diphosphate reductase (EC 1.17.4.1) has been studied with 19F-NMR. The product analogue is shown to be in fast exchange with substrate binding sites on protein subunit 1 (R1) of ribonucleoside-diphosphate (NDP) reductase. The number of binding sites is reduced to half when the complete holoenzyme R1R2 is formed. The temperature dependence of the line broadening of 5-FdUDP was studied using 19F-NMR, and of dCDP and dUDP using 1H-NMR. The temperature dependences are complex and a molecular model in which R1 is in a temperature dependent equilibrium between at least two conformations is suggested in order to explain the observed behaviour. Binding of a ligand to the substrate binding sites affects the conformational equilibrium in a ligand specific way. Formation of the holoenzyme R1R2 also affects the equilibrium.

Binding Sites↗
PubMed1985

Phosphatidylinositol hydrolysis and phosphatidylinositol 4',5-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas.

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca2+-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca2+ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of 32PO4 into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca2+-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This 32PO4 turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4',5'-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca2+ deficiency. This phosphatidylinositol 4',5'-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca2+ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4',5'-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4',5'-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca2+ mobilization.

Animals↗
PubMed1990

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays.

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.

Animals↗
PubMed2004

Synthesis and biological activity of isopentenyl diphosphate analogues.

A series of analogues of isopentenyl diphosphate (IPP) having a dicarboxylate moiety in place of the diphosphate were synthesized and investigated as inhibitors of undecaprenyl diphosphate (UPP) synthase and protein farnesyltransferase (PFTase). PFTase is involved in control of cell proliferation and is known to be inhibited by certain maleic acid derivatives bearing long alkyl substituents (> or =12 carbons, e.g., chaetomellic acid). UPP synthase is a potential target for antimicrobial agents and utilizes isopentenyl diphosphate (IPP) as a substrate. A number of dicarboxylate-containing IPP analogues were prepared in 2-5 steps from commercially available starting materials with good overall yield (20-78%). These syntheses involved the conjugate addition of an organocuprate to dimethyl acetylenedicarboxylate (DMAD) followed by basic ester hydrolysis. The E-pentenylbutanedioic acid 32 showed inhibition of UPP synthase with an IC(50) of 135 microM. Compound 30 displays competitive inhibition of PFTase with a K(i) of 287 microM.

Alkyl and Aryl Transferases↗
PubMed1998

Inhibitory effects of uridine diphosphate on UDP-glucuronosyltransferase.

Inhibitory effects of uridine diphosphate on the enzymatic activity of UDP-glucuronosyltransferase (UGT) were investigated. Pyrimidine nucleotides such as UDP, UTP and cytidine diphosphate reduced the activity of rat purified UGT (phenol UGT) to about 10%, 48% and 46% of the control, respectively, at the same concentration as a donor substrate, UDP-glucuronic acid. Purine nucleotides, uridine monophosphate, glucuronic acid and some UDP-sugars were only slightly inhibitory toward the transferase. Similar effects were observed in the expressed UGT (UGT1A6; corresponding to phenol UGT) in yeast cells and rat liver microsomal membrane-binding UGT, indicating that uracil and diphosphate residues are essential for the UDP inhibition. Interestingly, 2'-deoxy UDP was found to be a less effective inhibitor (about 50% inhibition) than UDP on the purified, the expressed (UGT1A6 and UGT2B1) and microsomal membrane-binding UGTs. These results indicate that not only uracil and diphosphate residues but also 2'-hydroxyl residue of UDP ribose participates in the interactions between UDP and UDP-glucuronosyltransferase.

Animals↗
PubMed2005

Role of arginine-304 in the diphosphate-triggered active site closure mechanism of trichodiene synthase.

The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP(i)) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PP(i) bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg2+(3)-PP(i), in which R304 donates hydrogen bonds to PP(i) and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP(i) in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP(i) and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg2+(3)-PP(i) binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP(i) leaving group.

Arginine↗
PubMed1997

Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase.

The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP and (+)-1-alpha,2-alpha,3-alpha-trihydroxy-4-beta-cyclopentanemethanol 5-phosphate, respectively, the binding of Mg2+ and the substrates were determined to occur via a steady state ordered mechanism in which Mg2+ binds to the enzyme first and ribose 5-phosphate binds last. Mg2+ binding to the enzyme prior to the binding of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands Mg x ATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate. Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by Mg x ADP indicated that Mg x ADP binding to the allosteric site may occur in competition with enzyme bound Mg2+. Ligand binding studies showed that 1 mol of Mg x ATP was bound per mol of phosphoribosyl diphosphate synthetase subunit, which indicated that the allosteric sites of the multimeric enzyme were not made up by inactive catalytic sites.

Adenosine↗
PubMed2000

Role of nucleoside diphosphate kinase in the activation of anti-HIV nucleoside analogs.

Nucleoside analogs are currently used in antiretrovirus therapies. The best known example is AZT one of the first drug to be used for the treatment of AIDS. However, only the triphosphate derivatives of these compounds act as substrates of the viral reverse transcriptase. Since they do not enter cells, nucleoside analogs are administered and phosphorylated by cellular kinases. The last step in this phosphorylation pathway is catalyzed by nucleoside diphosphate (NDP) kinase. The incorporation of the nucleoside triphosphates into nascent viral DNA chain results in termination of the elongation process. We have performed kinetics studies of the phosphorylation reaction by NDP kinase of dideoxynucleoside diphosphates such as 2',3'-dideoxy-3'-azidothymidine diphosphate (AZT-DP) and 2',3'-dideoxy-2',3'-didehydrothymidine diphosphate (d4T-DP). We show that the catalytic efficiency is strongly decreased and, therefore, that the reaction step catalyzed by NDP kinase constitutes a bottleneck in the processing pathway of anti-HIV compounds. In addition, the affinity of the analogs in the absence of catalysis was determined using a catalytically inactive NDP kinase mutant, showing a reduction of affinity by a factor of 2 to 30, depending on the analog. The structure of NDP kinase provides a structural explanation for these results. Indeed, all nucleoside analogs acting as chain terminators must lack a 3'-OH in the nucleotide deoxyribose. Unfortunately, this same substitution is detrimental for their capacity to be phosphorylated by NDP kinase. This defines the framework for the design of new nucleoside analogs with increased efficiency in antiretroviral therapies.

Anti-HIV Agents↗