The synthesis and properties in enzymic reactions of substrate analogs containing the methylphosphonyl group.

The properties of the methylphosphonyl group as a substrate analog for the phosphoryl moiety of various biological phosphoryl donors have been investigated in several enzymic phosphoryl transfer reactions. The synthesis and characterization of adenosine 5′-[β-methylphosphonyl]diphosphate, adenosine 5′-methylphosphonate, acetyl methylphosphonate, and methylphosphonoenolpyruvate are fully described. Adenosine 5′-[β-methylphosphonyl]diphosphate is not a substrate for adenylate kinase, hexokinase, 3-phosphoglycerate kinase, glycerol kinase, phosphofructokinase, creatine kinase, alkaline phosphatase, or nucleoside 5′-diphosphate kinase. Competitive inhibition of ATP was observed with hexokinase and 3-phosphoglycerate kinase with $\text{K}{}_{\mathrm{i}}\text{K}{}_{\mathrm{m}}\text{～- 10}$. Adenosine 5′-methylphosphonate was a substrate for adenylate deaminase and 5′-nucleotidase, but not for adenylate kinase, acid phosphatase, 5′-phosphodiesterase, or 3′-phosphodiesterase. Acetyl methylphosphonate inhibits the reaction of acetyl phosphate with acetate kinase, but methylphosphonoenolpyruvate has no effect upon the reaction of phosphoenolpyruvate with pyruvate kinase. The results indicate that with the exception of 5′-nucleotidase, the methyphosphonyl group is incapable of undergoing phosphoryl transfer. One interpretation among others is that a metaphosphate-type mechanism is required for these processes.     

Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures

Cycle-purified microtubule protein from mammalian brain incorporated [32P]Pi upon incubation with [gamma-32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [gamma-32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [gamma-32P]GTP to endogenous proteins followed a linear time-course and was stimulated by low concentrations of ATP and, more efficiently, by ADP. These data are in agreement with the predictions derived from a mechanism of phosphorylation by which [gamma-32P]GTP does not act as a phosphoryl donor for the protein kinase activity but, instead, only as a repository of high group transfer potential phosphoryl groups used to make [gamma-32P]ATP, from contaminating ADP, by means of the nucleoside diphosphate kinase activity. Using 100 mM fluoride, which suppressed protein phosphorylation without inhibiting the nucleoside diphosphate kinase activity, formation of [gamma-32P]ATP was detected. Fluoride was also able to protect microtubules from a slow depolymerization which was found to occur during long-term incubation of microtubules. This indicates that the phosphorylation observed in the presence of GTP is sufficient to destabilize microtubules.     

Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.     

Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation.

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.     

Phosphoryl transfer by a concerted reaction mechanism in UMP/CMP-kinase

The reaction mechanism of phosphoryl transfer catalyzed by UMP/CMP-kinase from Dictyostelium discoideum was investigated by semiempirical AM1 molecular orbital computations of an active site model system derived from crystal structures that contain a transition state analog or a bisubstrate inhibitor. The computational results suggest that the nucleoside monophosphate must be protonated for the forward reaction while it is unprotonated in the presence of aluminium fluoride, a popular transition state analog for phosphoryl transfer reactions. Furthermore, a compactification of the active site model system during the reaction and for the corresponding complex containing AlF3 was observed. For the active site residues that are part of the LID domain, conformational flexibility during the reaction proved to be crucial. On the basis of the calculations, a concerted phosphoryl transfer mechanism is suggested that involves the synchronous shift of a proton from the monophosphate to the transferred PO3-group. The proposed mechanism is thus analogous to the phosphoryl transfer mechanism in cAMP-dependent protein kinase that phosphorylates the hydroxyl groups of serine residues.     

Nucleoside diphosphate kinase from Xenopus oocytes; partial purification and characterization

We have identified and partially purified a soluble nucleoside diphosphate kinase (NDP kinase) from Xenopus laevis oocytes. The enzyme preparation can catalyze the transfer of phosphate from ATP to all of the major oxy- and deoxynucleotides. It can also catalyze the transfer of a phosphorothioate group from gamma-S-ATP to an acceptor GDP forming gamma-S-GTP. Like NDP kinases from other sources, the catalytic mechanism appears to involve a phosphoenzyme intermediate which can be isolated. Transfer of phosphate from nucleoside triphosphates to protein is rapid, reaching saturation within 1 min following the addition of nucleoside triphosphates. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. While nucleoside diphosphate kinases are generally thought to require magnesium for activity, both the oocyte enzyme preparation and a commercial bovine liver enzyme preparation are only partially inhibited by short (10 min) exposures to 25 mM EDTA. Both enzyme preparations are, however, further inhibited by long incubations with this metal chelator (2 h, 70% inhibition). Zinc enhances the inhibition of NDP kinase by EDTA, but is ineffective on its own. Rapid phosphorylation in the presence of [gamma-32P]ATP and EDTA could be used to identify the phosphoenzyme intermediate in homogenates of Xenopus oocytes and facilitated its isolation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with autoradiography indicated the presence of only a single phosphorylated species of Mr 21,500 in supernatants of fresh oocyte homogenates. Partial purification of this protein utilizing salt precipitation, hydrophobic-interaction chromatography and an affinity step with Affi-Gel Blue Sepharose resulted in a 100-fold purification and a 29% overall yield of NDP-kinase activity. Size-exclusion chromatography of the purified preparation yielded two peaks containing enzyme activity. They eluted with apparent molecular weights of 45,000 and 70,000, suggesting a native enzyme that is multimeric or associated with other proteins.     

Reverse and forward reactions of carbamyl phosphokinase from Streptococcus faecalis R. Participation of nucleotides and reaction mechanisms

The participation of Mg complex of nucleoside diphosphates and nucleoside triphosphates in the reverse and forward reactions catalyzed by purified carbamyl phosphokinase (ATP : carbamate phosphotransferase, EC 2.7.2.2) of Streptococcus faecalis R, ATCC-8043 were studied. The results of initial velocity studies of approx. 1 mM free Mg2+ concentration have indicated that in the reverse reaction MgdADP was as effective a substrate as MgADP. The phosphoryl group transfer from carbamyl phosphate to MgGDP, MgCDP and MgUDP was also observed at relatively higher concentrations of the enzyme and respective magnesium nucleoside diphosphate. In the forward direction MgdATP was found to be as efficient a phosphate donor as MgATP. On the other hand, Mg complexes of GTP, CTP and UTP were ineffective even at higher concentrations of the enzyme and respective magnesium nucleoside triphosphate. Product inhibition studies carried out at non-inhibitory level of approx. 1 mM free Mg2+ concentration have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate or nucleoside triphosphate and the other for carbamyl phosphate or carbamate, and its reaction with the substrates is of the random type. Further tests of numerical values for kinetic constants have indicated that they are partially consistent with the Haldane relationship which is characteristic of rapid equilibrium and random mechanism.     

The Human Nm23/Nucleoside Diphosphate Kinases

Biochemical experiments over the past 40 years have shown that nucleoside diphosphate(NDP) kinase activity, which catalyzes phosphoryl transfer from a nucleoside triphosphate toa nucleoside diphosphate, is ubiquitously found in organisms from bacteria to human. Overthe past 10 years, eight human genes of the nm23/NDP kinase family have been discoveredthat can be separated into two groups based on analysis of their sequences. In addition tocatalysis, which may not be exhibited by all isoforms, evidence for regulatory roles has comerecently from the discovery of the genes nm23 and awd, which encode NDP kinases and areinvolved in tumor metastasis and Drosophila development, respectively. Current work showsthat the human NDP kinase genes are differentially expressed in tissues and that their productsare targeted to different subcellular locations. This suggests that Nm23/NDP kinases possessdifferent, but specific, functions within the cell, depending on their localization. The roles ofNDP kinases in metabolic pathways and nucleic acid synthesis are discussed.     

Post-translational phosphorylation of serine 74 of human deoxycytidine kinase favors the enzyme adopting the open conformation making it competent for nucleoside binding and release

Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.     

Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase

The reaction mechanism of the catalytic phosphoryl transfer of cAMP-dependent protein kinase (cAPK) was investigated by semi-empirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol(-1) and the reaction itself to be exothermic by 12.2 kcal mol(-1). The active site residue Asp166, which was often proposed to act as a catalytic base, does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two-dimensional energy hypersurfaces of the protein kinase wild-type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hypersurface of this hypothetical mutant shows two possible pathways for phosphoryl transfer, which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.     

Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDP<-->ADP+(d)NTP. This reaction, suggested to occur by the transfer of the gamma-phosphoryl from ATP to the nucleoside diphosphate, is overall similar to that normally carried out by nucleoside diphosphate kinase (Ndk). Accordingly, Adk was proposed to be responsible for residual Ndk-like activity measured in a mutant strain of Escherichia coli, where the ndk gene was disrupted. We present data supporting a mechanism for the synthesis of nucleoside triphosphates by Adk that unlike the previously suggested mechanism mentioned above are in complete agreement with the current knowledge about the Adk enzyme and its various catalytic properties. We propose that nucleoside triphosphate synthesis occurs by beta-phosphoryl transfer from ADP to any bound nucleoside diphosphate. Our results point to the fact that the proposed Ndk-like mechanism of Adk originated from an erroneous interpretation of data, in that contamination of ATP preparations with AMP and ADP was not taken into account. Our results also address the proposed role of Adk in restoring a normal growth rate of mutant strains of E. coli lacking Ndk. These mutant strains apparently, in spite of a mutator phenotype, are able to synthesise nucleoside triphosphates by alternative pathways to maintain the same growth rate as the wildtype.     

The interaction of phosphorothioate analogues of ATP with phosphomevalonate kinase. Kinetic and 31P NMR studies

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma-bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor.     

Catalysis of the hydrolysis of phosphorylated pyridines by Mg(OH)+: a possible model for enzymatic phosphoryl transfer

The second-order rate constants for reaction of the Mg2+ complexes of phosphorylated pyridine monoanions with Mg(OH)+ are 10(4)-10(6)-fold larger than the second-order rate constants for their reaction with water (25 degrees C, ionic strength 1.5). Of the 10(6)-fold rate enhancement with the phosphorylated 4-morpholinopyridine/Mg2 complex, approximately 10(4)-fold is attributed to the greater nucleophilicity of Mg(OH)+ compared with water. The remaining catalysis of approximately 10(2)-fold is attributed to induced intramolecularity from positioning of the hydroxide ion and phosphoryl group by the Mg2+ ions. This reaction may provide a model for the role of a metal ion in increasing the concentration of the anions of enolpyruvate and serine and holding the nucleophile in the correct position for phosphoryl transfer in the reactions catalyzed by pyruvate kinase and alkaline phosphatase, for example. Some mechanisms that can provide catalysis of phosphoryl transfer through a metaphosphate-like transition state are reviewed briefly.     

Acetate kinase (pyrophosphate). A fourth pyrophosphate-dependent kinase from Entamoeba histolytica.

An enzyme from Entamoeba histolytica catalyzes the formation of acetyl phosphate and orthophosphate from acetate and inorganic pyrophosphate (PP_i), but it displays much greater activity in the direction of acetate formation. It has been purified 40-fold and separated from interfering enzyme activities by chromatography. Its reaction products have been quantitatively established. ATP cannot replace PP_i as phosphoryl donor in the direction of acetyl phosphate formation nor will any common nucleoside diphosphate replace orthophosphate as phosphoryl acceptor in the direction of acetate formation. The trivial name proposed for the new enzyme is acetate kinase (PP_i).     

Crystal structure of nucleoside diphosphate kinase required for coleoptile elongation in rice (Oryza sativa L.)

Nucleoside diphosphate kinase (NDK) is a ubiquitous enzyme found in all organisms and cell types, and catalyzes the transfer of the phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. The enzyme is involved in and required for coleoptile elongation in rice as the level of the rice NDK (rNDK) changes during seed germination and the early stages of seedling growth. The expression of rice NDK gene is up-regulated in the growing coleoptiles when the anaerobic stress persists. The rNDK structure determined at 2.5 A resolution consists of a four-stranded anti-parallel beta-sheet, of which the surfaces are partially covered with six alpha-helices; its overall and active site structures are similar to those of homologous enzymes except the major conformation variations of residue 132-138 regions, involving significant structural contacts. The model contains 148 residues of 149 residues in total and averaged 19 water molecules per monomer for 12 molecules in an asymmetric unit. A mold of 12 superimposed molecules shows that the alphaA-alpha2 area has greater variations and higher temperature factors, indicating the flexibility for a substrate entrance. Hexameric molecular packing in both crystal and solution implies that rNDK functions as hexamers. This rNDK structure, which is the first NDK structure from a higher plant system, provides the structural information essential to understand the functional significance of this enzyme during growth and development in both rice and other plants.     

Carbamate kinase of Lactobacillus buchneri NCDO110. II. Kinetic studies and reaction mechanism

The participation of Mg2+ or Mn2+ nucleoside diphosphates in the reverse reaction catalyzed by purified carbamate kinase (ATP:carbamate phosphotransferase, EC 2.7.2.2) of Lactobacillus buchneri NCDO110 was studied. The results of initial velocity studies have indicated that Mn2+ ADP is as effective as a substrate as Mg2+ ADP is. Product inhibition studies have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate and the other for carbamyl phosphate. The reaction of the enzyme with the substrates is of the random type.     

Functional Coupling between Nucleoside Diphosphate Kinase of the Outer Mitochondrial Compartment and Oxidative Phosphorylation

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.     

A simple method to purify ribonucleotide reductase

Assays of ribonucleotide reductase in extracts of Detroit 98 (human) cells were found to be complicated by the rapid depletion of the substrate (CDP) by nucleoside diphosphate kinase. Assays of either 100,000g supernatants or ammonium sulfate-fractionated extracts resulted in the conversion of >90% of the substrate to CTP within 2 min. It was therefore desirable to separate nucleoside diphosphate kinase from ribonucleotide reductase. Chromatography of the fractionated extract on an ATP-agarose column resulted in the delivery of nondissociated ribonucleotide reductase in the void volume and the retention of >99.9% of the nucleoside diphosphate kinase. The kinase could be eluted by 2 mm ATP. The ribonucleotide reductase was recovered from this commercially available gel with an apparent yield of >200%. It could be accurately assayed with only minimal extraneous depletion of substrate. Furthermore, it was stable to storage at ?80°C. Tris-HCl was found to inhibit the enzyme. When HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-Na buffer was used in place of Tris-HCl, the rate of CDP reduction was increased by 2.5-fold. Since the above procedure selectively removes nucleoside diphosphate kinase from crude preparations of ribonucleotide reductase, it should have general applicability for purifying ribonucleotide reductase from other sources.     

Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases

NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.     

Conformational changes during the catalytic cycle of gluconate kinase as revealed by X-ray crystallography

The crystal structure of gluconate kinase from Escherichia coli has been determined to 2.0 A resolution by X-ray crystallography. The three-dimensional structure was solved by multi-wavelength anomalous dispersion, using a crystal of selenomethionine-substituted enzyme. Gluconate kinase is an alpha/beta structure consisting of a twisted parallel beta-sheet surrounded by alpha-helices with overall topology similar to nucleoside monophosphate (NMP) kinases, such as adenylate kinase. In order to identify residues involved in substrate binding and catalysis, structures of binary complexes with ATP, the ATP analogue adenosine 5'-(beta,gamma-methylene) triphosphate and the product, gluconate-6-phosphate have been determined. Significant conformational changes are induced upon binding of ATP to the enzyme. The largest changes involve a hinge-bending motion of the NMP(bind) part and a motion of the LID with adjacent helices, which opens the cavity to the second substrate, gluconate. Opening of the active site cleft upon ATP binding is the opposite of what has been observed in the NMP kinase family so far, which usually close their active site to prevent fortuitous hydrolysis of ATP. The conformational change positions the side-chain of Arg120 to stack with the purine ring of ATP and the side-chain of Arg124 is shifted to interact with the alpha-phosphate in ATP, at the same time protecting ATP from solvent water. The beta and gamma-phosphate groups of ATP bind in the predicted P-loop. A conserved lysine side-chain interacts with the gamma-phosphate group, and might promote phosphoryl transfer. Gluconate-6-phosphate binds with its phosphate group in a similar position as the gamma-phosphate of ATP, consistent with inline phosphoryl transfer. The gluconate binding-pocket in GntK is located in a different position than the nucleoside binding-site usually found in NMP kinases.