Characterization of a nucleotide kinase encoded by bacteriophage t7

Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ～12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) m obtained with dTTP for dTMP kinase is ～3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) m), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.     

Regulation of human placental deoxyguanosine kinase by nucleotides

The activity of deoxyguanosine kinase purified from human placenta was regulated by various nucleotides. dTTP, an activator, only increased the Vmax value of the enzyme. The feedback inhibition by dGTP, dGDP and dGMP were competitive with respect to deoxyguanosine. Both the activation by dTTP and the inhibition by dGTP were reversible.     

Partial purification and characterization of deoxyguanosine kinase from pig skin.

Deoxyguanosine kinase, which catalyses the phosphorylation of deoxyguanosine to form deoxyguanosine 5'-monophosphate, was purified 1024-fold from extracts to newborn-pig skin. This activity requires the presence of a bivalent cation and a nucleoside triphosphate, which functions as a phosphate donor, ATP being twice as effective as CTP or GTP and 4 times as effective as UTP. The enzyme appears to have a molecular weight of 58500 as determined by Sephadex-column chromatography. Optimal enzymic activity was observed at pH 8.0; however, the enzyme remained active over a broad pH range of 5.5-9.0. Several deoxyribonucleoside and ribonucleoside monophosphates and triphosphates were tested as effectors of catalytic activity. Effective inhibitors were dGMP [Ki(app.) = 7.6 x 10(-5) M] and dGTP [Ki(app.) = 2.1 x 10(-5) M]. Both of these inhibitors acted in a competitive manner. A Km(app.) of 3.2 x 10(-7) M was measured for deoxyguanosine and a Km(app.) of 3.3 mM was determined for MgATP. Of the four major deoxynucleosides tested, this catalytic activity appears to phosphorylate only deoxyguanosine; thus the enzyme is a specific deoxyguanosine kinase.     

Properties of a highly purified mitochondrial deoxyguanosine kinase

Deoxyguanosine kinase, purified over 6000-fold from beef liver mitochondria by means of deoxyguanosine-3'-(4-aminophenyl phosphate)-Sepharose affinity chromatography, was nearly homogeneous. It phosphorylates only deoxyguanosine and deoxyinosine among the natural nucleosides, with apparent Km values of 4.7 and 21 microM, respectively. Among nucleoside analogs tested, only arabinosylguanine (Ki = 125 microM) and 8-aza-deoxyguanosine (Ki = 450 microM) competed with deoxyguanosine. The relative molecular mass of the enzyme is 56,000, as determined by equilibrium sedimentation, and sodium dodecyl sulfate-gel electrophoresis suggests two subunits of Mr 28,000. The pH optimum for enzyme activity is 5.5, but optimum enzyme stability is seen at pH 7.0. Triton X-100 increased the stability of the enzyme markedly. ATP is the best phosphate donor at pH 5.5, but pyrimidine triphosphates such as dTTP and UTP are more efficient donors at pH 7.4. The activation energy, at pH 5.5, was estimated to be 10.9 kcal/mol. Amino acid modification experiments suggest the involvement of arginine, cysteine, and probably histidine. The inactivation of the enzyme by modification of these amino acid residues was time and pH dependent. Both substrates protected the enzyme from inactivation in every case but that of photooxidation by Rose Bengal, where only deoxyguanosine prevented inactivation.     

The metabolism of deoxyguanosine in mitochondria: a characterization of the phosphorylation process which occurs in intact mitochondria

Isolated, intact mitochondria were evaluated for their ability to phosphorylate deoxyguanosine. This activity was stimulated by exogenous ATP, substrates for oxidative phosphorylation or added inorganic phosphate. Inhibitors of oxidative phosphorylation lowered the levels of deoxyguanosine phosphorylation. From a Hanes plot, an apparent Km of 0.83 microM deoxyguanosine was calculated for the phosphorylation activity in intact mitochondria. In the presence of a 20-fold excess of added deoxynucleosides, none of those tested were strongly inhibitory. However, added UDP and dTDP were stimulatory and dGTP and dGDP were inhibitory to the phosphorylation of deoxyguanosine. These data show that mitochondria phosphorylate deoxyguanosine and that the process is regulated by other events which take place within the organelle.     

Human thymidylate kinase. Purification, characterization, and kinetic behavior of the thymidylate kinase derived from chronic myelocytic leukemia.

Thymidylate kinase derived from the blast cells of human chronic myelocytic leukemia was purified 2186-fold to near homogeneity by means of alcohol precipitation, alumina-Cgamma gel fractionation, calcium phosphate gel fraction, ultrafiltration, and affinity column chromatography. The molecular weight was estimated by glycerol gradient centrifugation to be 50,000. This enzyme had an optimal activity at pH 7.1 and required a divalent cation in order to catalyze the reaction. Mg2+ and Mn2+ were found to be the preferential divalent cations. The activation energy was estimated to be 19.1 kcal/mol at pH 7.2. Initial velocity study suggested that the reaction followed a sequential mechanism. Mg2+ ATP had a Km of 0.25 mM and dTMP had a Km of 40 micrometer. The enzyme was unstable even at 4 degrees. In the presence of ATP or dTMP the enzyme maintained its activity. Purine triphosphate nucleosides were found to be better phosphate donors than the pyrimidine triphosphate nucleosides. ATP and dATP had a lower Km and a higher Vmax than GTP and dGTP. dTMP was the only preferred phosphate receptor among all the monophosphate nucleotides tested dTTP and IdUTP competed with both substrates and inhibited the reaction with a Ki of 0.75 mM and 1.1 mM, respectively.     

Phosphorylation of deoxyguanosine in intact and fractured mitochondria

The phosphorylation of deoxyguanosine was measured in fractured and intact mitochondria and an apparent K_m of 16 M for deoxyguanosine was calculated using fractured mitochondria. The effects of various deoxynucleotides on the phosphorylating activity in fractured organelles was tested at both a high and low ratio of NXP/ATP and at two pH values, 7.0 and 5.5. Exogenous dGTP, dGDP or dITP were inhibitory under all conditions tested. With a NXP/ATP ratio of 0.08 at pH 7.0, TTP, TDP, dADP, ADP, UTP and UDP were stimulatory, but at pH 5.5 only TTP elicited that response. When the NXP/ATP ratio was 10 at pH 5.5, TTP and UTP increased the activity more than 10-fold, whereas, at pH 7.0 TTP, TDP, dADP, ADP, UTP, UDP caused stimulation, but to a much lesser extent. When exogenous Mg²⁺, Mn²⁺ or Ca²⁺ were added to intact mitochondria, the rates of phosphorylation were lowered. In fractured mitochondria in the absence of exogenous ATP, little phosphorylation occurs, hence these metal ions caused little change. ATP-Mg, ATP-Mn and ATP-Ca, each at 0.05 mM caused a small inhibition with intact mitochondria, whereas, these compounds supported phosphorylation with fractured organelles. ATP-Mn (10 mM) or ATP-Ca (10 mM) stimulated phosphorylation in both intact and fractured mitochondria. Intact mitochondria synthesized dGMP, dGDP and dGTP when metal ion or ATP-Me concentrations were low (0.05 mM) or when Mg²⁺ concentration was high (10 mM). Additions of ATP-Ca, ATP-Mn, ATP-Mg, Mn²⁺ or Ca²⁺ at 10 mM cause the loss of dGDP and dGTP formation and, in most cases, an increase in the synthesis of dGMP. Fractured mitochondria make only dGMP and the levels of its synthesis are greater than that observed for intact mitochondria. These data suggest that intact mitochondria are required for the synthesis of dGTP and that its synthesis is regulated by mitochondria nucleotides.     

Deoxynucleoside kinases encoded by the yaaG and yaaF genes of Bacillus subtilis. Substrate specificity and kinetic analysis of deoxyguanosine kinase with UTP as the preferred phosphate donor

The overlapping yaaG and yaaF genes from Bacillus subtilis were cloned and overexpressed in Escherichia coli. Purification of the gene products showed that yaaG encoded a homodimeric deoxyguanosine kinase (dGK) and that yaaF encoded a homodimeric deoxynucleoside kinase capable of phosphorylating both deoxyadenosine and deoxycytidine. The latter was identical to a previously characterized dAdo/dCyd kinase (M?llgaard, H. (1980) J. Biol. Chem. 255, 8216-8220). The purified recombinant dGK was highly specific toward 6-oxopurine 2'-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2',3'-dideoxyguanosine. UTP was the preferred phosphate donor with a Km value of 6 microm compared with 36 microm for ATP. In addition, the Km for dGuo was 0.6 microm with UTP but 6.5 microm with ATP as phosphate donor. The combination of these two effects makes UTP over 50 times more efficient than ATP. Initial velocity and product inhibition studies indicated that the reaction with dGuo and UTP as substrates followed an Ordered Bi Bi reaction mechanism with UTP as the leading substrate and UDP the last product to leave. dGTP was a potent competitive inhibitor with respect to UTP. Above 30 microm of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.     

Human deoxythymidine kinase II: substrate specificity and kinetic behavior of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia.

Cytoplasmic and mitochondrial deoxythymidine kinase isozymes derived from the blast cells of acute myelocytic leukemia differ in their substrate specificity and kinetic behavior. These enzymes require divalent cations for their activity. The data suggest that the major role of idvalent cations is to chelate with ATP; the complex thus formed serves as the phosphate donor for the reaction. The activity of various triphosphate nucleosides as a phosphate donor for cytoplasmic deoxythymidine kinase is as follows: ATP = dATP greater than ara-ATP greater than GTP greater than CTP greater than dGTP = dCTP greater than dUTP, whereas for mitochondrial deoxythymidine kinase, the order of activity is ATP greater than CTP greater than UTP = dATP greater than ara-ATP greater than dGTP = dCTP greater than dUTP. Neither IdUTP nor dTTP could serve as a phosphate donor in the reaction catalyzed by either isozyme. From the many pyrimidine analogues tested for their binding affinity to each of these isozymes, I-dUrd and Br-dUrd had high good affinity which was equivalent to that of deoxythymidine. 5-Allyl-dUrd, 5-ethyl-dUrd, and 5-propyl-dUrd were only weakly bound to each isozyme. 5-I-dCyd, 5-Br-dCyd, dCyd, and 5-vinyl-dUrd were tightly bound to mitochondrial deoxythymidine kinase but not to the cytoplasmic isozyme. dTTP and I-dUTP are potent inhibitors of the reaction catalyzed by both isozymes. In contrast, dCTP and ara-CTP are potent inhibitors only of the mitochondrial isozyme, but not of the cytoplasmic isozyme. ATP-MG2+ acts as a sigmoidal substrate of the cytoplasmic isozyme with a"Km" of 0.22 mM, and as a regular substrate of the mitochondrial isozyme with a Km of 0.1 mM. Deoxythymidine acts as a regular substrate for both cytoplasmic and mitochondrial isozyme with a Km of 2.6 and 5.2 muM, respectively. Initial velocity as well as product inhibition studies suggest that the cytoplasmic isozyme catalyzes the reaction via a "sequential" mechanism. In contrast, mitochondrial deoxythymidine kinase catalyzes the reaction via a "ping-pong" mechanism.     

Saccharomyces cerevisiae nucleoside-diphosphate kinase: purification, characterization, and substrate specificity.

Nucleoside-diphosphate kinase is an enzyme which catalyzes the phosphorylation of nucleoside diphosphates into the corresponding triphosphates for nucleic acid biosynthesis. In this communication, we describe the purification and characterization of nucleoside-diphosphate kinase from yeast. The purified protein appears to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis, with a molecular weight of about 17,000-18,000. An estimate from the fast protein liquid chromatography Superose 12 gel filtration shows a native molecular weight of about 68,000 to 70,000. The results suggest that yeast nucleoside-diphosphate kinase is composed of four subunits. Substrate specificity studies show that the relative activity of nucleoside diphosphates (NDP) as phosphate acceptors is in the order of dTDP greater than CDP greater than UDP greater than dUDP greater than GDP greater than or equal to dGDP greater than dCDP greater than dADP greater than ADP; and the relative activity of triphosphate donors is in the order of UTP greater than dTTP greater than CTP greater than dCTP greater than dATP greater than ATP greater than or equal to dGTP greater than GTP. The Km and Vm of dTDP, dGDP, dCDP, dUDP, CDP, and UDP have been determined. The rate constant studies indicate that the purified NDP kinase prefers using, to a slight extent, dTDP (approximately 800 min-1) as the substrate rather than other tested deoxyribo- and ribonucleotides (350-450 min-1). The broad substrate specificity and kinetic data suggest that the enzyme is involved in both DNA and RNA metabolism.     

The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of purine-nucleoside phosphorylase deficiency

Purine-nucleoside phosphorylase (PNP) is a purine degradative enzyme that catalyzes the phosphorolysis of (deoxy) inosine or (deoxy) guanosine to their respective bases and (deoxy) ribose 1-phosphate. A severe T-cell immune deficiency syndrome with hypouricemia is associated with impaired PNP function. To study the biochemical basis for this syndrome we created an in vitro model of PNP deficiency in mitogen (phytohemagglutinin)-stimulated normal human peripheral blood lymphocytes using guanosine to competitively inhibit deoxyguanosine phosphorolysis. Guanosine-induced guanine toxicity was reversed by adenine. Under these conditions, deoxyguanosine (5-45 microM) diminished mitogen stimulation to 30% of control while increasing the deoxyguanosine triphosphate pool (dGTP) by over 20-fold. Deoxycytidine reversed deoxyguanosine toxicity with a diminution of dGTP accumulation, but no significant change in the deoxycytidine triphosphate pool. Thymidine reversed the deoxyguanosine toxicity, repleted the thymidine triphosphate (dTTP) pool, and caused an even further increase in the accumulation of dGTP. These data support a model of lymphotoxicity in PNP deficiency based on dGTP accumulation with inhibition of ribonucleotide reductase and depletion of the thymidine triphosphate pool. Thymidine triphosphate depletion is reversed by either deoxycytidine or thymidine; however, the former diminishes dGTP accumulation (probably by competition for phosphorylation) and the latter potentiates dGTP accumulation (probably through feedback augmentation of guanosine diphosphate (GDP) reduction by ribonucleotide reductase secondary to an increased dTTP pool).     

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.     

Biosynthesis of deoxynucleoside triphosphates,dCTP and dTTP: reaction mechanism and kinetics

The enzyme reaction mechanism and kinetics for biosyntheses of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) from the corresponding deoxycytidine diphosphate (dCDP) and deoxythymidine diphosphate (dTDP) catalyzed by pyruvate kinase were studied. The kinetic model for the two synthetic reactions was found to follow the Bi–Bi random rapid equilibrium mechanism similar to that of the biosynthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP). Kinetic constants involved in the reactions including the maximum reaction velocity, the Michaelis–Menten constants, and the inhibition constants for dCTP and dTTP biosyntheses were experimentally determined. This enzyme reaction requires Mg²⁺ ion and the optimal Mg²⁺ concentration was also determined. The experimental results showed a good agreement with the simulation results obtained from the kinetic model developed. The kinetics of the four biosynthetic reactions for deoxynucleoside triphosphates (dNTP) including dATP, dGTP, dCTP, and dTTP from the corresponding deoxynucleoside diphosphates (dNDP) including dADP, dGDP, dCDP, and dTDP were analyzed. The results suggest that the binding kinetics of phosphoenolpyruvate (PEP) and pyruvate are similar for all four biosynthetic reactions. The affinity of the dNDP substrates to enzyme is of the same order of magnitude as the corresponding dNTP as inhibitors. The order of reactivity and substrate specificity for dNDP is dADP > dGDP > dCDP > dTDP in the pyruvate kinase (PK) reactions. The results obtained from this study can be applied to bioreactor design and production of dCTP and dTTP for biosynthesis of DNA at a significantly lower cost compared to the currently available chemical method.     

The metabolism of deoxyguanosine in mitochondria: relationship of the uptake of deoxyguanosine to the electron transport chain and adenosine triphosphate

That deoxyguanosine is taken up by isolated rat liver mitochondria has been shown. This report describes the relationship between that uptake and oxidative phosphorylation. By measuring this process in the presence of standard inhibitors of oxidative phosphorylation it was determined that a functional electron transport chain, but not the phosphorylation of ADP, was essential for uptake. ATP analogs adenyl(beta,gamma-methylene)diphosphate and adenylimidodiphosphate blocked uptake, indicating that ATP hydrolysis was required. ADP also proved to be an inhibitor. Exogenous UTP slightly stimulated deoxyguanosine uptake, as did added ATP, but several other nucleotides (dGTP, dATP, UDP, dGDP) were inhibitory. A sulfhydryl group was important for deoxyguanosine uptake and inhibition of uptake by N-ethylmaleimide was protected by both deoxyguanosine and ATP. These data show that deoxyguanosine uptake by mitochondria is a process which is coordinated and, perhaps, regulated by other events which take place in the organelle.     

Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides

Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.     

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.     

Identification and characterization of a unique adenosine kinase from Mycobacterium tuberculosis

Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.     

Nucleoside 5'-diphosphates as effectors of mammalian ribonucleotide reductase

It was found that nucleoside 5'-diphosphates could serve as effectors of ribonucleotide reductase. ADP was an activator of CDP reduction; ADP reduction was activated by dGDP; GDP reduction was activated by dTDP. Conversely, dADP inhibited the reduction of CDP, UDP, GDP, and ADP; dGDP inhibited UDP and GDP reductions; and dTDP inhibited UDP reduction. The inhibition of UDP reduction by dADP, dTDP, and dGDP was at least equal to that observed for dATP, dTTP, and dGTP, respectively. In these experiments with the nucleoside diphosphates as effectors, high-pressure liquid chromatography analysis of the reaction mixtures showed that no nucleoside 5'-triphosphates were found during the reaction period which could account for the effects seen with the nucleoside diphosphates as effectors. Further experiments were carried out in which adenyl-5'-yl imidodiphosphate was used as the positive effector of CDP and UDP reductions in place of ATP. Under these conditions, CDP and UDP reductions were inhibited by dADP, dTDP, and dGDP to the same extent observed in the presence of ATP. ADP served not only as a substrate for ribonucleotide reductase but also as an activator of CDP and UDP reductions. The direct products (dNDPs) also served as positive and negative effectors. Dixon plots indicated that the dNDPs were acting as noncompetitive inhibitors with respect to the substrate. ADP increased the sedimentation velocity of the ribonucleotide reductase in a manner similar to ATP. These data are consistent with the allosteric effects seen with the nucleoside 5'-triphosphates. Additionally, from the thorough study of the role of effectors on UDP reduction, it is clear that UDP reduction was most sensitive to the negative effectors dATP, dADP, dTTP, dTDP, dGTP, and dGDP.     

Adenosine kinase from human liver

Adenosine kinase (ATP: adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to homogeneity from human liver. The yield was 55% of the initial activity with a final specific activity of 6.3 mumol/min per mg protein. The molecular weight was estimated as about 40 000 by Sephadex G-100 gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 18% of that of adenosine. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad optimum at pH 7.5--8.5. The Km value for adenosine was 0.15 micrometer, and the activity was strongly inhibited at higher concentrations than 0.5 micrometer. ATP, dATP, GTP and dGTP were proved to be effective phosphate donors. Co2+ was more effective than Mg2+, and Ca2+, Mn2+, Fe2+ and Ni2+ showed about 50% of the activity for Mg2+. Some difference in structure between the adenosine kinase from human liver and that from rabbit or rat tissue, was observed by amino acid analysis and peptide mapping analysis.     

The soluble adenosine triphosphatase of Thiobacillus ferrooxidans.

Adenosine triphosphatase (ATPase) from Thiobacillus ferrooxidans was purified 55-fold. Polyacrylamide gel electrophoresis of the most purified fraction showed only one major band; histochemical analysis showed that the ATPase activity was associated with this band. The pH optimum is 9-10. The enzyme hydrolyzed ATP stoichiometrically to ADP and inorganic phosphate, the Km for this substrate being 7.75 times 10-3 M. GTP and ITP are alternate substrates, the Km values for these being 6.71 times 10-3 M and 3.12 times 10-3 M, respectively. ADP is slightly hydrolyzed. Magnesium, manganese, and calcium can serve as cofactors; Km values for these are 2.0 times 10-3 M, 9.4 times 10-4 M, and 8.0 times 10-4 M, respectively. The enzyme activity was not activated by either sodium or potassium, but a combination of the two ions were inhibitory. Azide and p-hydroxymercuribenzoate strongly inhibited the enzyme activity, whereas cyanide, dinitrophenol, and N,N'-dicyclohexylcarbodiimide (DCCD) were without effect. The enzyme was cold labile at 0 degrees-C, but was more stable at 18-24 degrees-C.