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Nucleoside diphosphate kinases catalyze the reversible transfer of the gamma phosphate of nucleoside triphosphates to nucleoside diphosphates. This minireview presents recent advances in understanding the reaction mechanism using steady-state and fast kinetic studies, X-ray crystallography, and site-directed mutagenesis. We also briefly discuss the physiological relevance of in vitro studies.
The abnormal wing discs gene of Drosophila encodes a soluble protein with nucleoside diphosphate kinase activity. This enzymic activity is necessary for the biological function of the abnormal wing discs gene product. Complete loss of function, i.e., null, mutations cause lethality after the larval stage. Most larval organs in such null mutant larvae appear to be normal, but the imaginal discs are small and incapable of normal differentiation. Killer-of-prune is a neomorphic mutation in the abnormal wing discs gene. It causes dominant lethality in larvae that lack prune gene activity. The Killer-of-prune mutant protein may have altered substrate specificity. Null mutant larvae have a low level of nucleoside diphosphate kinase activity. This suggests that there may be additional Drosophila genes that encode proteins with nucleoside dipthosphate kinase activity. Candidate genes have been found in the Drosophila genome.
PURPOSE: This study is concerned with cellular delivery/generation of 2'-azido-2'-deoxyuridine and -deoxycytidine diphosphate (N3UDP or N3CDP), potent inhibitors of ribonucleotide reductase. It characterizes the phosphorylation steps involved in the conversion of 2'-azido-2'-deoxyuridine (N3Urd) and 2'-azido-2'-deoxycytidine (N3Cyd) to the corresponding diphosphates and explores a prodrug approach in cellular delivery of the inhibitor which circumvents the requirement of deoxynucleoside kinases. METHODS: Cell growth of CHO and 3T6 cells of known deoxycytidine kinase level was determined in the presence of N3Urd and N3Cyd. Activity of ribonucleotide reductase was determined in the presence of the azidonucleosides as well as their mono- or di-phosphates in a Tween 80-containing permeabilizing buffer. A prodrug of 5'-monophosphate of N3Urd was prepared and its biological activity was evaluated with CHO cells as well as with cells transfected with deoxycytidine kinase. RESULTS: N3Urd failed to inhibit the growth of both cell lines, while N3Cyd was active against 3T6 cells and moderately active against CHO cells. These results correlate with the deoxycytidine kinase levels found in the cells. Importance of the kinase was further established with the finding that the nucleoside analogs were inactive as reductase inhibitors in a permeabilized cell assay system while their mono- and di-phosphates were equally active. The prodrug was active in cell growth inhibition regardless of the deoxycytidine kinase level. CONCLUSIONS: The azidonucleosides become potent inhibitors of the reductase by two sequential phosphorylation steps. The present study indicates that the first step to monophosphate is rate-limiting, justifying a prodrug approach with the monophosphate.
1. Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were investigated by use of patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, cell-attached configuration and inside-out patches). 2. Levcromakalim produced a concentration-dependent outward current at a holding potential of -50 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch configurations. 3. In conventional whole-cell configuration, the levcromakalim (100 microM)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-induced outward current still remained after 18 min. 4. The peak amplitude of the levcromakalim (100 microM)-induced outward membrane current recorded by the conventional whole-cell configuration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but significant outward membrane current remaining was abolished by glibenclamide. 5. In conventional whole-cell recordings, inclusion of an NDP in the pipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamide. Addition of 100 microM levcromakalim after the NDP-induced current had peaked activated a further outward current which was larger than that recorded in the absence of NDPs. Approximately 50% of this current still remained at 18 min, even when conventional whole-cell configuration was used. 6. In the cell-attached mode in symmetrical 140 mM K+ conditions, glibenclamide inhibited the 100 microM levcromakalim-activated 43 pS K+ channel in a concentration-dependent manner, showing an inhibitory dissociation constant (Ki) of approximately 520 nM. 7. In inside-out patches in which the glibenclamide-sensitive K+ channel had run down after exposure to levcromakalim, both uridine 5'-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application of Mg2+ to the UDP-reactivated K+ channels enhanced the channel activity reversibly. 8. In inside-out patches UDP was capable of activating the glibenclamide-sensitive K+ channel without levcromakalim, providing that there was free Mg2+ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg2+). Additional application of levcromakalim caused a further reversible activation of channel opening. 9. In the presence of levcromakalim, application of adenosine 5'-triphosphate (ATP) to the inner surface of the membrane patch inhibited UDP-reactivated channel opening in a concentration-dependent manner. 10. Addition of an untreated cytosolic extract of pig proximal urethra reactivated the glibenclamide-sensitive K+ channel in the presence of 100 microM levcromakalim in inside-out patches. 11. These results demonstrate the presence in the pig proximal urethra of a glibenclamide-sensitive K+ channel that is blocked by intracellular ATP and can be activated by levcromakalim. Intracellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg2+ may play an important role in regulating the channel activity.
Adenosine 5'-diphosphate (ADP) induces human blood platelets to aggregate and change shape, and it has been suggested that these two responses are mediated by more than one subtype of ADP receptor. The structure-activity relationships for several analogues of adenine nucleotides in causing aggregation and shape change were measured and compared in washed platelets using an aggregometer. ADP and its analogues 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP), adenosine 5'(alpha,beta-methylene)diphosphonate (AMPCP), S(P)-adenosine 5'-O-(1-thiodiphosphate) (AD-P alphaS) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) were used as agonists. Adenosine 5'-triphosphate (ATP) and its analogues, P1, P5-diadenosine pentaphosphate (ApsA), adenosine (5'-(alpha,beta-methylene)triphosphonate (AMPCPP), 2-methylthioadenosine 5'-triphosphate (2-methylthio-ATP) and uridine 5'-triphosphate (UTP), as well as the trypanocidal drug suramin, were used as antagonists. In general, the structure-activity relationships for both responses were similar, but for some analogues differences were observed. ADPalphaS and ADPbetaS were much more potent agonists relative to ADP for shape change than for aggregation and indeed ADPalphaS antagonized ADP-induced aggregation with an apparent pK(B) value of 5.5+/-0.1. 2-Methylthio-ATP also had different effects in aggregation and shape change, being a much higher affinity antagonist of aggregation than of shape change with an apparent pK(B) value of 7.0+/-0.2 for aggregation and 5.2+/-0.2 for shape change. These results support the suggestion that these two responses are mediated by multiple ADP receptors on human platelets, and are consistent with shape change being mediated via one receptor (the P2Y1 receptor) with aggregation requiring the activation of two receptors (the P2Y1 and another P2Y receptor).
1. Receptor-independent activation of heterotrimeric G proteins by plasma membrane-associated nucleoside diphosphate kinase (NDPK) has been demonstrated in vivo, and elevated levels of NDPK were found in purified sarcolemmal membranes of patients with end-stage heart failure. 2. Among 22 consecutive patients with chronic heart failure who underwent cardiac transplantation, those treated with a beta-blocker (n=8) had a 65% lower NDPK content and activity in the cardiac sarcolemma, compared to patients with similar base line characteristics who had no beta-blocker therapy (n=14). 3. The lower NDPK was associated with a reduced NDPK-dependent, Gi-mediated inhibition of adenylyl cyclase activity, as assessed by in vitro measurement of adenylyl cyclase activity in the presence of GDP or its kinase-resistant analog guanosine 5'-O-(2-thio)diphosphate (GDPbetaS). 4. We further tested whether treatment with a beta-adrenergic agonist would induce an increase in sarcolemmal NDPK. Rats treated with isoproterenol developed myocardial hypertrophy, and NDPK in the sarcolemma rose by 60% during 14 days of treatment. The beta-blocker propranolol prevented both effects. When hypertrophy was induced with thyroid hormone, NDPK did not increase. 5. In conclusion, chronic activation of beta-adrenergic receptors increases the binding of NDPK to cardiac sarcolemma, where it may activate heterotrimeric G proteins.
Ak (adenylate kinase) is a ubiquitous enzyme that catalyses a reversible high-energy phosphoryl-transfer reaction between ATP and AMP to form ADP. In the present study, the Ak gene (adk) of Mycobacterium tuberculosis was cloned, expressed in Escherichia coli and purified as a glutathione S-transferase fusion protein. Purified Ak converted AMP into ADP in the presence of [gamma-32P]ATP or [gamma-32P]GTP. Replacement of arginine-88 of adk with glycine resulted in the loss of enzymic activity. The purified protein also showed Ndk (nucleoside diphosphate kinase)-like activity as it transferred terminal phosphate from [gamma-32P]ATP to all nucleoside diphosphates, converting them into corresponding triphosphates. However, Ndk-like activity of Ak was not observed with [gamma-32P]GTP. Immunoblot analysis of various cellular fractions of M. tuberculosis H37Rv revealed that Ak is a cytoplasmic protein. The dual activity of Ak as both nucleoside mono- and di-phosphate kinases suggested that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism.
The transfer, catalysed by pig liver microsomal preparations, of mannose, from GDP-mannose, to lipid-linked oligosaccharides and the properties of the products are described. Solubility, hydrolytic and chromatographic data suggest that they are dolichol diphosphate derivatives. The presence of two N-acetyl groups in at least part of the heterogenous oligosaccharide portion was tentatively deduced. Reduction with borohydride of the oligosaccharide showed that the newly added mannose residues were not at its reducing end. Periodate oxidation suggested that 60% of these were at the non-reducing terminus and that 40% were positioned internally. T.l.c. showed the presence of seven oligosaccharide fractions with chromatographic mobilities corresponding to glucose oligomers with 7-13 residues. The molar proportions of the oligosaccharide fractions in the mixture were determined by borotritiide reduction and the number of mannose residues added to each oligosaccharide fraction during the incubation was calculated. Two of the oligosaccharide fractions had received on average one, or slightly more than one, mannose residue per chain during the incubation; four of the other fractions were each shown to be a mixture, 20-25% of which had received one mannose residue during the incubation and 75-80% of which had not been mannosylated during the incubation. This supported other evidence for the presence of endogenous lipid-linked oligosaccharides in the microsomal preparation which had been formed before the incubation in vitro. Evidence for the possibility of two pools of dolichol monophosphate mannose, one being more closely associated with mannosyl transfer to dolichol diphosphate oligosaccharides than the other, is also discussed.
Nucleoside diphosphate kinase (NDPK) is a highly conserved multifunctional enzyme. It catalyses the transfer of gamma phosphates from nucleoside triphosphates to nucleoside diphosphates by a mechanism that involves formation of an autophosphorylated enzyme intermediate. The phosphate is usually supplied by ATP. NDPK activity in different subcellular compartments may regulate the crucial balance between ATP and GTP or other nucleoside triphosphates. NDPKs are homo-oligomeric proteins and are predominantly localized in the cytosol. In this paper, we demonstrate that in Saccharomyces cerevisiae a small fraction of total NDPK activity encoded by YNK1 is present in the intermembrane space (IMS) of mitochondria, and the corresponding protein Ynk1p in the IMS represents approx. 0.005% of total mitochondrial proteins. Ynk1p, synthesized as a single gene product, must therefore be partitioned between cytoplasm and mitochondrial IMS fractions. A mechanism for this partitioning is suggested by our observations that interaction with a 40 kDa protein of the translocase of outer mitochondrial membrane (Tom40p), occurs preferentially with unfolded, unphosphorylated forms of Ynk1p. A population of newly translated, but not yet folded or autophosphorylated, Ynk1p intermediates may be imported into the IMS of mitochondria and trapped there by subsequent folding and oligomerization. Within the small volume of the IMS, Ynk1p may be more concentrated and may be required to supply GTP to several important proteins in this compartment.
1. Incubation of human platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) causes a time-dependent increase in the activation of adenylate cyclase due to Gs (the stimulatory GTP-binding protein). Forskolin enhances adenylate cyclase activity but does not interfere with the process of activation. The activation follows first-order kinetics in both the presence and the absence of the assay components. 2. ATP in the presence or the absence of an ATP-regenerating system of phosphocreatine and creatine kinase inhibits activation. 3. Hydrolysis of ATP to ADP does not lead to receptor-mediated inhibition of adenylate cyclase acting via Gi (the inhibitory GTP-binding protein). The ADP analogue adenosine 5'-[beta-thio]diphosphate (ADP[S]) does not inhibit the activation process. 4. Phosphocreatine alone inhibits adenylate cyclase activation at concentrations above 1 mM. 5. Inhibition by phosphocreatine is not due to the chelation of free Mg2+ ions. 6. Inhibition by ATP and the other assay components occurs throughout the activation process, decreasing both the rate of activation and the maximum activity obtained. 7. Maximal activation of adenylate cyclase after prolonged incubation with p[NH]ppG slowly reverses in the presence of the assay components. 8. A 10-fold excess of the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]) over p[NH]ppG inhibits the activation process completely, at all stages of the time course. 9. Preincubations in the presence and absence of ATP, cyclic AMP, phosphocreatine and creatine kinase show equal sensitivity to increasing GDP[S] concentration. These data show that the inhibition observed in the presence of ATP is not due to endogenous or contaminating guanine nucleotides, and suggest that phosphoryl transfer may regulate adenylate cyclase activity.
Nucleoside diphosphate kinase (NDP kinase) catalyses the phosphate transfer between nucleoside triphosphates and nucleoside diphosphates. As formation of guanosine triphosphate could be dependent on ATP in neutrophils, the presence of NDP kinase was tested in these phagocytic cells. Both membrane and cytosolic fractions of human neutrophils were found to contain NDP kinase activity. The specific activity measured in the cytosol appeared 10-fold higher than in the membrane and was not modified when the cells were activated with phorbol 12-myristate 13-acetate. Interestingly, stimulation with N-formylmethionyl leucylphenylalanine in the presence of cytochalasin B showed an increase in membrane NDP kinase activity together with the translocation of the enzyme from the cytosol to the membrane, suggesting a possible role of NDP kinase in regulating G-proteins as previously reported. In addition, activation with opsonized zymosan induced an increase in cytosolic activity, suggesting different regulation depending on the signal transduction pathway. The neutrophil enzyme consisted of two subunits of 21 kDa (NDPKA) and 18 kDa (NDPKB) again essentially present in the cytosol of the cell. Separation of proteins by two-dimensional PAGE demonstrated that each subunit consisted of at least four isoforms, indicating post translational modifications. A characteristic of this family of enzymes is the stability of the phosphorylated intermediate. In neutrophils, only one acidic isoform of each NDPKA and NDPKB was labelled in the presence of EDTA. In addition, non-denatured complexes were apparent between 91 and 130 kDa, suggesting a hexameric structure as was also proposed for NDP kinases from other eukaryotic cells. These complexes were found to differ in their isoelectric points, indicating the existence of various isoenzymes probably resulting from combination between several isoforms of each subunit.
We report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis. The N-terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli. immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4 B matrix and passing the total cell extract through it revealed four proteins (P70, P65, P60, and P50, respectively) of M(r) 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of M(r) 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P70 protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.
Nucleoside diphosphate kinase (Ndk) is an important enzyme that generates nucleoside triphosphates (NTPs) or their deoxy derivatives by terminal phosphotransfer from an NTP such as ATP or GTP to any nucleoside diphosphate or its deoxy derivative. As NTPs, particularly GTP, are important for cellular macromolecular synthesis and signalling mechanisms, Ndk plays an important role in bacterial growth, signal transduction and pathogenicity. Specific examples of the role of Ndk in regulating growth, NTP formation and cell surface polysaccharide synthesis in two respiratory tract pathogens, Pseudomonas aeruginosa and Mycobacterium tuberculosis, are discussed.
Nucleoside diphosphate (NDP) kinase phosphorylates nucleoside diphosphates with little specificity for the base and the sugar. Although nucleotide analogues used in antiviral therapies are also metabolized to their triphosphate form by NDP kinase, their lack of the 3'-hydroxyl of the ribose, which allows them to be DNA chain terminators, severely impairs the catalytic efficiency of NDP kinase. We have analyzed the kinetics parameters of several mutant NDP kinases modified on residues (Lys16, Tyr56, Asn119) interacting with the gamma-phosphate and/or the 3'-OH of the Mg2+-ATP substrate. We compared the relative contributions of the active-site residues and the substrate 3'-OH for point mutations on Lys16, Tyr56 and Asn119. Analysis of additional data from pH profiles identify the ionization state of these residues in the enzyme active form. X-ray structure of K16A mutant NDP kinase shows no detectable rearrangement of the residues of the active site.
Escherichia coli nucleoside diphosphate kinase (Ndk) catalyzes ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the cognate diphosphate precursor. Recently, the Ndk polypeptide was reported to be a multifunctional base excision repair nuclease that processed uracil residues in DNA by acting sequentially as a uracil-DNA glycosylase inhibitor protein (Ugi)-sensitive uracil-DNA glycosylase, an apurinic/apyrimidiniclyase, and a 3'-phosphodiesterase [Postel, E. H. & Abramczyk, B. M. (2003) Proc. Natl. Acad. Sci. USA 100, 13247-13252]. Here we demonstrate that the E. coli Ndk polypeptide lacked detectable uracil-DNA glycosylase activity and, hence, was incapable of acting as a uracil-processing DNA repair nuclease. This finding was based on the following observations: (i) uracil-DNA glycosylase activity did not copurify with Ndk activity; (ii) Ndk purified from E. coli ung(-) cells showed no detectable uracil-DNA glycosylase activity; and (iii) Ndk failed to bind to a Ugi-Sepharose affinity column that tightly bound E. coli uracil-DNA glycosylase (Ung). Collectively, these observations demonstrate that the E. coli Ndk polypeptide does not possess inherent uracil-DNA glycosylase activity.
Escherichia coli nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations. It is also one of several recently discovered DNases belonging to the NM23/NDK family. E. coli cells disrupted in the ndk gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases. Another explanation for the increased mutation rates is that endk- cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway. Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA. We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: (i) uracil-DNA glycosylase that excises uracil from single-stranded and from U/A and U/G mispairs in double-stranded DNA; (ii) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and (iii) DNA repair phosphodiesterase that removes 3'-blocking residues from the ends of duplex DNA. All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein. Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA.
The phosphate transfer system of Haseltine et al., consisting of a ribosomal wash obtained from a stringent strain of Escherichia coli, washed ribosomes, GTP, and ATP, was used to prepare large quantities of guanosine tetra- and pentaphosphates, the magic spot compounds MS I and MS II of Cashel and Gallant. In our hands, the Haseltine et al. system yielded predominantly guanosine tetraphosphate, ppGpp. This system was used exclusively in the described experiments, with ATP labeled with (32)P in the beta- and gamma-positions as donor. The beta-label was found to produce a ppGp⃰p and the gamma-label a ppGpp⃰. Furthermore, [(3)H]GDP + [gamma-(32)P]ATP yielded ppGpp in a (3)H:(32)P ratio of 1:1. The results indicate a transfer of the terminal pyrophosphoryl group of ATP as a unit. The position of the transferred pyrophosphoryl was assayed for by preparation of pGp⃰ from ppGp⃰p with Zn(++)-activated inorganic pyrophosphatase from yeast. The pGp⃰ was then assayed with 3'-nucleotidase, which liberated practically all the labeled phosphate. This result indicate that the phosphate transfer from ATP to GDP yields guanosine 5'-diphosphate-3'-diphosphate.
Purified calf thymus ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1), showed an absolute requirement for a dithiol as hydrogen donor, whereas the natural monothiol glutathione (GSH) was inactive per se. However, a protein partially purified from thymus coupled the oxidation of GSH to the formation of deoxyribonucleotides by ribonucleotide reductase. In analogy with the ribonucleotide reductase system of Escherichia coli this protein was called glutaredoxin [Holmgren, A. (1976) Proc. Natl. Acad. Sci. USA 73, 2275-2279]. Thymus glutaredoxin had the following properties: (i) its molecular weight determined by gel chromatography was about 12,000; (ii) it was active iwth ribonucleotide reductase in the presence of GSH, NADPH, and glutathione reductase but had no activity with NADPH and thioredoxin reductase; and (iii) it was immunologically different from thioredoxin because it did not bind to antithioredoxin immunoadsorbents. Experiments on the crossreactivity of thymus and E. coli ribonucleotide reductases and the corresponding thioredoxin and glutaredoxin systems showed essentially no specificity for the homologous thioredoxin but a high species specificity for the homologous glutaredoxin.