Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human,and the Drosophila melanogaster,deoxyribonucleoside kinases

In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP). With dCK and ATP1, products were shown to be 5'-phosphates. With dCK, donor properties of the analogues were dependent on the nature of the acceptor, as with natural 5'-triphosphate donors. With dCK (dCyd as acceptor), Km and Vmax for the two 2'(3')-deoxyadenosine-3'(2')-triphosphates are similar to those for ATP. With dGK, Km values are higher than for ATP, while Vmax values are comparable. Kinetic studies further demonstrated Michaelis-Menten (non-cooperative) or cooperative kinetics, dependent on the enzyme employed and the nature of the donor. The physiological significance, if any, of the foregoing remains to be elucidated. The overall results are, on the other hand, highly relevant to studies on the modes of interaction of nucleoside kinases with donors and acceptors; and, in particular, to interpretations of the recently reported crystal structures of dGK with bound ATP, of dNK with bound dCyd, and associated modeling studies.     

Inorganic tripolyphosphate (PPP(i)) as a phosphate donor for human deoxyribonucleoside kinases

Inorganic tripolyphosphate (PPP(i)) and pyrophosphate (PP(i)) were examined as potential phosphate donors for human deoxynucleoside kinase (dCK), deoxyguanosine kinase (dGK), cytosolic thymidine kinase (TK1), mitochondrial TK2, and the deoxynucleoside kinase (dNK) from Drosophila melanogaster. PPP(i) proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not dAdo, as acceptor substrate, illustrating also the dependence of donor properties on acceptor. Products of phosphorylation were shown to be 5(')-phosphates. In striking contrast to ATP, the phosphorylation reaction follows strict Michaelis-Menten kinetics, with K(m) values of 74 and 92 microM for dCK and dGK, respectively, and V(max) values 40-50% that for ATP. With the other three enzymes, as well as for dCK with dAdo as acceptor, no, or only low levels (相似文献   

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.     

Ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all human and Drosphila melanogaster deoxyribonucleoside kinases

Six non-conventional adenosine-2'- and 3'-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5'-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

Non-allosteric regulation of the uridine kinase from seeds of Zea mays.

Uridine kinase (ATP: uridine 5'-phosphotransferase, EC 2.7.1.48) has been partially purified from ungerminated hybrid corn seed. It is associated with a soluble high molecular weight fraction from which it apparently cannot be dissociated without loss of activity. The stability of the enzyme is enhanced by the addition of dithiothreitol, glycerol and nucleotide substrate. The nucleoside specificity of the enzyme is limited to nucleosides containing pyrimidine and ribose moieties, such as uridine and cytidine. High concentrations of nucleosides cause substrate inhibition, however. The Km values for uridine and cytidine are 53 muM and 125 muM, respectively, and under subsaturating conditions uridine is phosphorylated about five times faster than cytidine. The reaction follows an ordered Bi Bi kinetic pattern, with ATP and ADP in competition for the free form of the enzyme. Purine, but not pyrimidine, nucleoside triphosphates serve as phosphate donors without regard to the sugar moiety. However, all of these triphosphates appear to compete for the same site on the enzyme. (Km ATP equals 590 muM, Km (app) GTP equals 61 muM, and CTP and UTP are linear competitive inhibitors against ATP, with Ki values of 60 muM and 240 muM, respectively.) Therefore, end product control of uridine kinase apparently does not involve allosteric sites, but instead is envisioned as simple competition between relatively effective or ineffective phosphate donors for a position on the enzyme.     

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.     

Ability of Adenosine-2′(3′)-deoxy-3′(2′)-triphosphates and Related Analogues to Replace ATP as Phosphate Donor for all Human and Drosphila melanogaster Deoxyribonucleoside Kinases

Abstract

Six non-conventional adenosine-2′- and 3′-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3′-deoxyadenosine-2′-triphosphate was an effective donor (5–60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5′-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

Multisubstrate analogs for deoxynucleoside kinases. Triphosphate end products and synthetic bisubstrate analogs exhibit identical modes of binding and are useful probes for distinguishing kinetic mechanisms

Comparative inhibition kinetics with natural dNTP end products (dNp3) and new synthetic bisubstrate-type analogs, dNp4A (deoxynucleoside 5'-adenosine 5'-P1,P4-tetraphosphate), have been studied with their target deoxynucleoside kinases from Lactobacillus acidophilus. Analysis of inhibition specificity, inhibition patterns, and Ki(app) under various conditions has revealed the following conclusions. Both dNTP and dNp4A bind to the active site of the corresponding kinase through multiple binding determinants. The deoxynucleoside moiety of dNTP fits optimally at the deoxynucleoside binding site and provides the basis for its inhibition specificity, whereas the triphosphate group interacts with the ATP binding site, reinforcing the affinity of the molecule as a potent end product inhibitor (Ki = 0.4-3 microM). The adenosine moiety of dNp4A does not contribute to the binding of this compound, whereas the tetraphosphate portion is the second binding determinant, just as in the model developed for dNTP. dNTP and dNp4A proved to be useful tools for distinguishing the kinetic mechanisms of kinases which follow sequential pathways, i.e. the rapid equilibrium Random Bi Bi for dCyd and dGuo kinases and the steady state Ordered Bi Bi mechanism for two dAdo kinases associated either with dCyd kinase or with dGuo kinase on different multifunctional proteins.     

Distinct sites for deoxyguanosine and deoxyadenosine phosphorylation on a monomeric kinase from Lactobacillus acidophilus

Base-line separation of two paired deoxynucleoside kinase activities (deoxycytidine/deoxyadenosine and deoxyguanosine/deoxyadenosine kinase), previously resolved as overlapping peaks from Blue Sepharose, has now been achieved. The improved separation and recovery in relatively small volumes were accomplished by eluting Blue Sepharose with a bisubstrate mixture: 0.5 mM dCyd plus 1 mM ATP released dCyd/dAdo kinase, and 1 mM dGuo plus 5 mM ATP eluted dGuo/dAdo kinase. The latter pair of activities showed copurification through UDP-Sepharose affinity chromatography and HPLC anion-exchange chromatography. The HPLC preparation appeared to be homogeneous, on the basis of nondenaturing polyacrylamide gel electrophoresis at several gel concentrations and pH values. Both dGuo and dAdo kinase activities coincided with the protein band. A single band of protein was also observed upon sodium dodecyl sulfate gel electrophoresis. The estimated molecular weight of the denatured protein (56 000) agrees closely with values obtained for native activity by sedimentation equilibrium or gel permeation chromatography. The rate of dAdo phosphorylation was found to be stimulated more than 3-fold by the presence of dGuo, and dGuo kinase was also slightly activated by the presence of dAdo. This mutual activation indicates that dGuo and dAdo kinase activities do not share a common site. Selective chemical inactivation of dGuo kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine eliminated the ability of dGuo to stimulate dAdo kinase in parallel with the loss of dGuo kinase activity. These lines of evidence strongly suggest that dGuo and dAdo kinase activities are functions of separate sites on a monomeric polypeptide and that these sites may be in allosteric communication.     

End-product regulation and kinetic mechanism of guanosine-inosine kinase from Escherichia coli

Escherichia coli guanosine-inosine kinase was overproduced, purified, and characterized. The native and subunit molecular weights were 85,000 and 45,000, respectively, indicating that the enzyme was a dimer. A pI of 6.0 was obtained by isoelectric focusing. In addition to ATP, it was found that deoxyadenosine 5'-triphosphate, UTP, and CTP could serve as phosphate donors. The phosphate acceptors were guanosine, inosine, deoxyguanosine and xanthosine, but not adenosine, cytidine, uridine, or deoxythymidine. Maximum activity was attained at an ATP/Mg2+ concentration ratio of 0.5. In the presence of pyrimidine nucleotides, enzyme activity was slightly increased, while it was markedly inhibited by GDP and GTP. Initial velocity and product inhibition studies support an ordered Bi Bi mechanism in which guanosine was the first substrate to bind and GMP was the last product to be released. Guanosine kinase may be a regulatory enzyme that has a role in modulating nucleotide levels.     

Mutations within the putative active site of heterodimeric deoxyguanosine kinase block the allosteric activation of the deoxyadenosine kinase subunit

Replacement of the Asp-84 residue of the deoxyguanosine kinase subunit of the tandem deoxyadenosine kinase/ deoxyguanosine kinase (dAK/dGK) from Lactobacillus acidophilus R-26 by Ala, Asn, or Glu produced increased Km values for deoxyguanosine on dGK. However, it did not seem to affect the binding of Mg-ATP. The Asp-84 dGK replacements had no apparent effect on the binding of deoxyadenosine by dAK. However, the mutant dGKs were no longer inhibited by dGTP, normally a potent distal endproduct inhibitor of dGK. Moreover, the allosteric activation of dAK activity by dGTP or dGuo was lost in the modified heterodimeric dAK/dGK enzyme. Therefore, it seems very likely that Asp-84 participates in dGuo binding at the active site of the dGK subunit of dAK/dGK from Lactobacillus acidophilus R-26.     

Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.     

Kinetic mechanism of the type II calmodulin-dependent protein kinase: studies of the forward and reverse reactions and observation of apparent rapid-equilibrium ordered binding

The kinetic reaction mechanism of the type II calmodulin-dependent protein kinase was studied by using its constitutively active kinase domain. Lacking regulatory features, the catalytic domain simplified data collection, analysis, and interpretation. To further facilitate this study, a synthetic peptide was used as the kinase substrate. Initial velocity measurements of the forward reaction were consistent with a sequential mechanism. The patterns of product and dead-end inhibition studies best fit an ordered Bi Bi kinetic mechanism with ATP binding first to the enzyme, followed by binding of the peptide substrate. Initial-rate patterns of the reverse reaction of the kinase suggested a rapid-equilibrium mechanism with obligatory ordered binding of ADP prior to the phosphopeptide substrate; however, this apparent rapid-equilibrium ordered mechanism was contrary to the observed inhibition by the phosphopeptide which is not supposed to bind to the kinase in the absence of ADP. Inspection of product inhibition patterns of the phosphopeptide with both ATP and peptide revealed that an ordered Bi Bi mechanism can show initial-rate patterns of a rapid-equilibrium ordered system when a Michaelis constant for phosphopeptide, Kip, is large relative to the concentration of phosphopeptide used. Thus, the results of this study show an ordered Bi Bi mechanism with nucleotide binding first in both directions of the kinase reaction. All the kinetic constants in the forward and reverse directions and the Keq of the kinase reaction are reported herein. To provide theoretical bases and diagnostic aid for mechanisms that can give rise to typical rapid-equilibrium ordered kinetic patterns, a discussion on various sequential cases is presented in the Appendix.     

Human deoxycytidine kinase: kinetic mechanism and end product regulation

The kinetic properties of the monomeric deoxycytidine kinase (EC 2.7.1.74) from leukemic human T-lymphoblasts have been investigated. The results of steady-state initial-rate kinetic analysis and product inhibition studies at pH 7.5 and 37 degrees C indicate that substrate binding follows an ordered sequential pathway, with the magnesium salt of ATP being the first substrate to bind and dCMP the last product to dissociate. At subsaturating substrate concentrations, dCMP produced competitive inhibition against ATP, while against varied deoxycytidine concentrations dCMP exhibited mixed-type inhibition. ADP produced noncompetitive inhibition against either substrate. The limiting Km values for deoxycytidine and MgATP were 0.94 and 30 microM, respectively. The end product inhibitor dCTP exhibited competitive inhibition against varied ATP concentration, with a dissociation constant estimated to be 0.7 microM when extrapolated to zero ATP concentration. dCTP was purely noncompetitive against varied deoxycytidine concentration. On the basis of these kinetic results, and on the strong and specific inhibition by dCTP, it is proposed that this end product functions as a multisubstrate analogue, with its triphosphate group binding to the phosphate donor site of the enzyme and its deoxycytidine moiety overlapping and binding to the deoxynucleoside site in a highly specific manner.     

Multisubstrate analogs for deoxynucleoside kinases. Use in novel affinity media applied to resolution of Lactobacillus enzymes

New multisubstrate-type inhibitors of the deoxynucleoside kinases have been synthesized, tested for their specificity as soluble inhibitors of enzymes from Lactobacillus acidophilus, and used to construct media for affinity chromatography. Each inhibitor was a deoxynucleoside 5'-adenosine 5"'-P1,P4-tetraphosphate (abbreviated dNp4A, where dN represents a dAdo, dCyd, dGuo, or dThd moiety linked through its 5'-hydroxyl to the terminal phosphate of adenosine tetraphosphate). At micromolar concentrations, each inhibitor strongly and specifically inhibited the corresponding deoxynucleoside kinase. Each of the four Lactobacillus deoxynucleoside kinase activities was selectively retained on its homologous dNp4A-Sepharose affinity medium. The activity was eluted on addition of the respective dNp4A with up to 70% recovery and 300-500-fold purification (relative to an ammonium sulfate fraction). Whereas dThd kinase was retained only by the dTp4A column, a portion of the dAdo kinase activity was retained, along with all the dCyd kinase or dGuo kinase, on dCp4A- or dGp4A-Sepharose, respectively, and coeluted with these activities. Conversely, all three activities were quantitatively retained on dAp4A-Sepharose, without competition from either dCyd or dGuo, and were eluted simultaneously upon addition of dAp4A. These observations further confirm the understanding that this organism employs paired, and presumably bifunctional, kinases, namely dCyd/dAdo kinase and dGuo/dAdo kinase, along with a separate thymidine kinase.     

Phosphorylation of isocarbostyril- and difluorophenyl-nucleoside thymidine mimics by the human deoxynucleoside kinases

The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-beta-D-ribofuranosyl)-isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were <1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1-(2-deoxy-beta-D-ribofuranosyl)-7-iodoisocarbostyril (EN2) showed poor relative-phosphorylation efficiencies (kcat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluoro-5-iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-beta-L-ribofuranosyl)]-aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.     

Partial purification and properties of rat-heart adenosine kinase.

The activity of myocardial adenosine kinase (E.N. 2.7.1.20) in a number of species was assayed. Rat heart contained the highest specific activity. From this source adenosine kinase was purified in a simple way 80-fold, until it was free of adenosine deaminase activity. A molecular weight of about 39 000 was measured. NSC 113939 (1), NSC 113940 and 8-azaadenosine inhibited myocardial adenosine kinase. Dipyridamole stimulated the enzyme at high adenosine levels, and inhibited at low substrate concentrations. A number of divalent cations could (partially) substitute for Mg2+. The optimal concentration of MgCl2 or MnCl2 was about 0.5 mM; concentrations exceeding 1 mM inhibited severely. An apparent Km for ATP of 0.1 mM was measured, whereas an apparent Km for adenosine of 0.5 muM was was found. The latter increased to 3.3 muM, when dipyridamole was added. Replacement of ATP by GTB or ITP increased the activity, and UTP and CTP were inferior as a phosphate donor.     

Reverse reaction of smooth muscle myosin light chain kinase. Formation of ATP from phosphorylated light chain plus ADP

Incubation of smooth muscle phosphorylated heavy meromyosin in the presence of myosin light chain kinase, calmodulin, ADP, and Ca2+ results in a decrease of the protein-bound phosphate. The dephosphorylation is not due to phosphatase activity and is dependent on the presence of ADP and the active ternary myosin light chain kinase complex. Using 32P-labeled phosphorylated 20,000-dalton light chains as the phosphate donor, the formation of ATP from ADP can be demonstrated. This reaction requires the presence of Ca2+, calmodulin, and myosin light chain kinase. These results indicate that myosin light chain kinase can catalyze a reverse reaction and form ATP from ADP and phosphorylated substrate. The rate of the reverse reaction, kcat/KLC approximately 0.21 min-1 microM-1, is considerably slower than the forward reaction under similar conditions and is therefore detectable only at relatively high concentrations of myosin light chain kinase. For the reverse reaction, KmADP is approximately 30 microM and ATP is a competitive inhibitor, KIATP approximately 88 microM. For the forward reaction, measured with both isolated light chains and intact myosin, KmATP is approximately 100 microM and ADP is a competitive inhibitor, KiADP approximately 140 microM (myosin) and 120 microM (light chains). Thus, the affinity of ATP for the forward and reverse reactions is similar, but the affinity of ADP is higher for the reverse reaction. From the light chain dependence of the two reactions, the following was calculated: forward, Km = 5 microM, kcat = 1720 min-1, and reverse, Km = 130 microM, kcat = 27 min-1. In contrast to the data obtained with isolated light chains, it is suggested that, with intact myosin as substrate, the Km term is primarily responsible for determining the rate of the reverse reaction. With light chains phosphorylated at serine 19 and threonine 18, it was shown that both sites act as a phosphate donor, although the reverse reaction for threonine 18 is slower than that for serine 19.     

Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus

Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein-serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+-calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond.     

Glycerol metabolism in higher plants: glycerol kinase

Glycerol kinase activity was identified in extracts of higher plant seeds and seedlings, and was partially purified and characterized from cucumber radicle tissue. The enzyme was localized in the post-mitochondrial supernatant of the cell, and catalyzed the formation of glycerol-3-phosphate. The pH optiumum was 9.0. ATP, CTP, GTP or UTP could be used as the phosphoryl group donor. The Km for glycerol was 55 microM and Km values for the nucleoside triphosphates were 145-620 microM. The Vmax for the reaction was 40-78 pmol product per min. Kinetic data indicate that the enzyme has a sequential mechanism.