Catalytic properties of Escherichia coli polyphosphate kinase: an enzyme for ATP regeneration.

Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.     

Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies

Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods. Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP. In addition, the Michaelis constants differ greatly from the substrate dissociation constants. Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme. Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies. Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site. The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides. The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities. Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities. The possible physiological importance of the regulatory behavior with respect to ATP is considered.     

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.     

Adenylate kinase activity in Mycobacterium leprae

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) was detected in partially purified preparations of cell-free extracts of Mycobacterium leprae. The apparent Km values of M. leprae adenylate kinase for ADP and Mg2+ were 1 X 10(-4) M, respectively. The enzyme was heat-labile: loss of activity by 80% at 45 degrees C and over 90% at 60 degrees C occurred within 5 min. M. leprae adenylate kinase was distinct from armadillo adenylate kinase in respect of affinity for substrate and heat-sensitivity.     

Adenylate kinase of plants. Properties of adenylate kinase of pea leaves]

The properties of adenylate kinase in 2 ADP in equilibrium ATP + AMP reaction have been studied. The dependence of the enzyme activity on medium pH, protein concentration, substrates, Mg++ ions, AMP, adenine and adenosine has been also investigated. pH optimum is found to be 8.5 for forward reaction and 8-9--for the reverse one. The Michaelis constants are as follows: for ADP--1.17-10(-4) M, for ATP--3.33-10(-4) M at 24 degrees C, in 50 mM tris-HCl pH 7.6. The optimal ratio, Mg++ ions/substrates (ADP, ATP + AMP), is 1:2. The chelates of adenine nucleotides with Mg++ ions are proved to be "true" reaction substrates. Unlike adenine and adenosine, the product of AMP reaction inhibits adenylate kinase activity. It is concluded that the properties of adenylate kinase in plants are similar to those of animals and humans (moikinase).     

Coenzyme production using immobilized enzymes. I. Preparation, characterization, and laboratory-scale application of an immobilized NAD kinase

NAD⁺ kinase (ATP: NAD⁺ 2-phosphotransferase, EC2.7.1.23) isolated from chicken liver was immobilized on a silica-based support possessing aldehyde functional groups. The highest catalytic activity achieved was 16 U g⁻¹ solid. The optimal pH for the catalytic activity of the immobilized NAD⁺ kinase was pH 7.1–7.3. The apparent optimum temperature for the immobilized enzyme was about 5°C higher than that of the soluble enzyme. There were no significant differences in the K_{m app} values. The immobilization improved the conformational stability of the enzyme. In preliminary experiments, a 95% conversion of NAD⁺ to NADP⁺ was achieved with use of the immobilized NAD⁺ kinase, which preserved its starting activity practically unchanged up to 36 days.     

High level expression of chicken muscle adenylate kinase in Escherichia coli

Chicken muscle adenylate kinase was produced in a large amount in Escherichia coli cells harboring an expression plasmid, pKK-cAKl-1. The plasmid was constructed by placing the cDNA sequence for chicken muscle adenylate kinase after the tac promoter. After induction by isopropyl-beta-D-thiogalactopyranoside, the enzyme protein amounted to about 10% of the bacterial proteins. The enzyme was readily purified in two steps by using phosphocellulose and Sephadex G-100 columns. The apparent molecular weight of the enzyme produced in E. coli was estimated to be 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in agreement with the value deduced from the cDNA sequence. Ten amino acids in the NH2-terminal region were determined, and were identical with the sequence deduced from the cDNA sequence except that the terminal methionine was absent. Michaelis constants for ATP, ADP, and AMP of the enzyme thus synthesized were essentially identical to those determined with the enzyme in crude extracts of chicken skeletal muscle.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Polyphosphate kinase activity in yeast vacuoles]

The enzyme polyphosphate kinase (ATP: Polyphosphate phosphotransferase EC 2.7.4.1) relating to the class of transferases was detected in the vacuoles of Saccharomyces carlsbergensis yeast. The direct ATP: Polyphosphate phosphotransferase reaction resulting in the synthesis of polyphosphates from ATP was shown to occur mainly in vacuoles. The localization of the reverse polyphosphate: ADP phosphatransferase reaction was not established in any of the subcellular yeast fractions studied. The activity of the direct reaction in the yeast protoplasts makes up about 1% of the reverse one, but in vacuoles it is significantly higher and makes up to 19%. Under activation of biochemical processes involved in the production of cell wall components by protoplasts, vacuolar polyphosphates work mainly in the direction of ATP synthesis at the expense of polyphosphates accumulated in vacuoles.     

Identification,characterization of levoglucosan kinase,and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

The first enzyme responsible for assimilating levoglucosan in Aspergillus niger CBX-209 was corroborated to be levoglucosan kinase that catalyzes the transfer of a phosphate group from ATP to levoglucosan to yield a glucose 6-phosphate in the presence of magnesium ion and ATP by FAB-mass spectrometric method combined with previous observations from HPLC and enzymological experiments. Levoglucosan kinase was purified to apparent homogeneity by using a combination of seven purification steps. SDS-PAGE revealed a single protein band of 56 KDa. It is a monomeric enzyme and maximal enzyme activity was measured at pH 9.3 and 30 degrees C. This kinase is stable below 20 degrees C at a quite broad pHs ranging from 6 to 10 and levoglucosan could protect the enzyme from thermal inactivation. Exclusive substrate specificity for levoglucosan suggested that not only the structure of the intramolecular glucosidic linkage but also the configuration of the pyranose frame would be specific for recognition by levoglucosan kinase. The K(m) values of this enzyme were 71.2mM for levoglucosan and 0.25 mM for ATP, determined by double reciprocal plottings and ADP inhibited on the enzyme activity competitively with a Ki value of 0.20mM. A cDNA library from A. niger was constructed in Escherichia coli DH5alpha. The library was screened for levoglucosan kinase gene on NCE selective medium and three positive recombinants were selected after a five day culture. Detection of activities of levoglucosan kinase in the cell extracts indicated that levoglucosan kinase gene (lgk) was expressed by the recombinant strain of E. coli DH5alpha.     

Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm

Two isozymes of creatine kinase have been purified from sperm of the sea urchin, Strongylocentrotus purpuratus. One isozyme was purified from the sperm flagellum, and the other from the head. Both require nonionic detergent for extraction from sperm. The flagellar isozyme is a monomeric species with an Mr of 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126,000 from sucrose density gradient and gel filtration analyses. Creatine kinase from sperm heads was localized to the mitochondrion by an antibody raised against mouse muscle creatine kinase. This purified mitochondrial isozyme is multimeric, with an Mr of 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but 240,000 for the native enzyme. Peptide mapping indicates that the two isozymes are not related. The following kinetic characteristics were observed for the purified flagellar and mitochondrial isozymes, respectively. In the direction of ATP formation, at pH 6.6 and 25 degrees C, specific activities were 235 and 180 units/mg; pH optima were 6.7 and 6.9 and Michaelis constants were 0.13 and 0.055 mM for ADP and 5.8 and 2.7 mM for phosphocreatine. In the direction of phosphocreatine formation, at pH 7.5 and 25 degrees C, specific activities were 29 and 47 units/mg; pH optima were 7.5 and 7.7 and Michaelis constants were 0.89 and 0.31 mM for ATP and 39 and 62 mM for creatine. These unique isozymes constitute the termini of the phosphocreatine shuttle of sea urchin sperm that is responsible for energy transport from the mitochondrion to the distal flagellum (Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334; Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys. J., 52, 75-86).     

Cloning, expression, isolation and properties of thymidine kinase herpes simplex virus, strain L2

A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.     

Purification and properties of squid mantle adenylate kinase. Role of NADH in control of the enzyme.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) from the mantle muscle of the squid, Loligo pealeii, was purified over 170-fold to homogeneity as judged by polyacrylamide and starch gel electrophoresis. The tissue contains a single isozyme of adenylate kinase, the enzyme from cytoplasmic and mitochondrial compartments (90 and 10% of total activity, respectively) being identical in physical and kinetic properties. Molecular weight was found to be 27,000 +/- 400. The enzyme shows a pH optimum of 8.2 in the forward (APD utilizing) and 7.4 in the reverse direction. Michaelis constants for ADP, ATP, and AMP are 0.70, 0.13, and 0.15 mM, respectively, with optimal Mg2+:adenylate ratios being 1:2 for ADP and 1:1 for ATP. A comparison of mass action ratios with the equilibrium constant indicated that squid adenylate kinase is held out of equilibrium in resting, but not active, muscle. A search for metabolic modulators of adenylate kinase revealed that NADH (Ki of 0.1 mM) was the only modulator which exerted a significant effect within its in vivo concentration range. The data presented indicate that NADH inhibition is the factor maintaining adenylate kinase in a nonequilibrium state in resting muscle and that release of this inhibition can serve to integrate adenylate kinase into the known scheme of intermediary metabolism in this tissue. A sharp drop in NADH levels at the onset on muscular work co-ordinates that activation of aerobic metabolism in this tissue and allows adenylate kinase to return to equilibrium function. At equilibrium, the enzyme can function to ampligy the concentration of AMP, a potent activator and deinhibitor of key glycolytic and Krebs cycle enzymes. The effect of modulators of adenylate kinase in preventing denaturation by heat or proteolysis revealed that NADH and substrates induced conformational changes in the enzyme which rendered it less susceptible to denaturation. The conformation state induced by NADH differed from that induced by substrate.     

Characterization of the thymidine kinase of Physarum polycephalum

Thymidine kinase [ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21] has been purified more than 3,500 fold from microplasmodia of Physarum polycephalum. Properties of the enzyme were determined on preparations purified 1,400 fold. Thymidine was transformed to dTMP while a stoichiometric quantity of ATP was transformed to ADP. 5-Iododeoxyuridine, 5-bromodeoxyuridine, and 5-fluorodeoxyuridine acted as competitive inhibitors for the thymidine substrate while 5-bromodeoxyuridine could be used as a substrate. In contrast uridine did not inhibit the enzymatic activity while deoxyuridine was a very poor competitive inhibitor in agreement with the observation that deoxyuridine could not be used as a substrate. Two apparent Michaelis constants were found for thymidine. Only the highest Michaelis constant could be decreased in the presence of increasing concentrations of ATP. Among the various nucleoside mono, di, or triphosphates studied only ATP and to a less extent dATP could be used as phosphate donors. A non competitive inhibition for thymidine was observed with dTTP. dTMP, dTDP, and dTTP acted as competitive inhibitors for ATP. None of the nucleoside mono, di, or triphosphates studied showed an activatory effect at low concentrations of ATP, even in the presence of dTTP. However, dUTP and dGDP acted as competitive inhibitors for ATP.     

Immobilized glutamate oxaloacetate transaminase. Steady state kinetic analysis and stability studies.

1. Glutamate oxaloacetate transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) was immobilized on amino ethyl cellulose using the bifunctional reagent diethyl adipimidate. 2. The steady state kinetic analysis was performed for the particulate and the free enzyme, and the Michaelis constants measured for the amino ethyl cellulose derivative were not greatly different from those measured for the free glutamate oxaloacetate transaminase, while the latter were in good agreement with values in the literature. 3. The amino ethyl cellulose-glutamate oxaloacetate transaminase was slightly more stable than the free enzyme at 65 degrees C, but was stabilised less by polyethylene glycol than the free enzyme.     

Investigation of the properties of bovine heart creatine kinase cross-linked with dimethyl suberimidate

Dimeric bovine heart creatine kinase (EC 2.7.3.2, ATP: creatine N-phosphotransferase) has been cross-linked with the bifunctional reagent dimethyl suberimidate at several concentrations to yield modified enzyme with enhanced stability towards heat denaturation. The degree of thermal stability is dependent on the degree of cross-linking with optimal stabilization occurring when approx. half of all the available amino groups are covalently attached to dimethyl suberimidate. Accelerated storage studies were performed and the results used to predict the storage time of the native and modified enzyme at lower temperatures. The cross-linked derivative was predicted to have a longer shelf-life at 4 degrees C than the native enzyme. Modification caused a reduction in the specific activity of the enzyme. The pH profile was altered following cross-linking, but the Michaelis constants were not changed. The modified enzyme exhibited a marked resistance to the action of some denaturing agents.     

Enzymes of adenylate metabolism and their role in hibernation of the white-tailed prairie dog, Cynomys leucurus

AMP deaminase (AMPD) and adenylate kinase (AK) were purified from skeletal muscle of the white-tailed prairie dog, Cynomus leucurus, and enzyme properties were assayed at temperatures characteristic of euthermia (37 degrees C) and hibernation (5 degrees C) to analyze their role in adenylate metabolism during hibernation. Total adenylates decreased in muscle of torpid individuals from 6.97 +/- 0. 31 to 4.66 +/- 0.58 micromol/g of wet weight due to a significant drop in ATP but ADP, AMP, IMP, and energy charge were unchanged. The affinity of prairie dog AMPD for AMP was not affected by temperature and did not differ from that of rabbit muscle AMPD, used for comparison. However, both prairie dog and rabbit AMPD showed much stronger inhibition by ions and GTP at 5 degrees C, versus 37 degrees C, and inhibition by inorganic phosphate, NH(4)Cl, and (NH(4))(2)SO(4) was much stronger at 5 degrees C for the prairie dog enzyme. Furthermore, ATP and ADP, which activated AMPD at 37 degrees C, were strong inhibitors of prairie dog AMPD at 5 degrees C, with I(50) values of 1 and 14 microM, respectively. ATP also inhibited rabbit AMPD at 5 degrees C (I(50) = 103 microM). Strong inhibition of AMPD at 5 degrees C by several effectors suggests that enzyme function is specifically suppressed in muscle of hibernating animals. By contrast, AK showed properties that would maintain or even enhance its function at low temperature. K(m) values for substrates (ATP, ADP, AMP) decreased with decreasing temperature, the change in K(m) ATP paralleling the decrease in muscle ATP concentration. AK inhibition by ions was also reduced at 5 degrees C. The data suggest that adenylate degradation via AMPD is blocked during hibernation but that AK maintains its function in stabilizing energy charge.     

Effect of adenine nucleotides on the reaction catalyzed by pyruvate,orthophosphate dikinase in maize

The effect of adenine nucleotides in pyruvate, orthophosphate dikinase (EC 2.7.9.1, ATP, pyruvate, orthophosphate phosphotransferase)_was studied with the enzyme furified from maize, and with the enzyme obtained from mesophyll chloroplast extracts during assay in the direction of pyruvate conversion to phosphoenolpyruvate. (1) In studies with the purified enzyme, the relationship of initial velocity to ATP concentrations follows Michaelis-Menten kinetics, and the K_m value for ATP was 22.8 μM (± 5.1 μM, n = 5). (2) AMP was a competitive inhibitor with respect to ATP, and its K_i value was 35.8 μM (± μM, n = 4). There was no inhibition of catalysis by ADP up to a concentration of 460 μM. (3) The theoretical response of the enzyme to change in the adenylate energy charge was calculated from the kinetic constants for ATP and AMP. The experimentally obtained values were similar to the theoretical response when varying energy charge was generated by addition of appropriate amounts of ATP, ADP and AMP in assays with the purified enzyme. The response of the enzyme to energy charge at different pH values (pH 7.0, 7.5, and 8.0) was similar, although the activity of the enzyme at pH 7.0 was about 40% of that at pH 8.0. (4) When mesophyll chloroplast extracts of maize, which contain high levels of adenylate kinase, were used as the source of the enzyme and the adenylate energy charge was generated by addition of different concentrations of ATP and AMP, the influence on catalysis was similar to that with the purified enzyme. (5) The data show that the effect of varying energy chage on the activity of the dikinase is not typical of a U-type enzyme, in contrast to phosphoglycerate kinase (EC 2.7.2.3, ATP: 3-phospho-D-glycerate 1-phosphotransferase), which is more strongly regulated. (6) Evidence is presented for competition between the dikinase and phosphoglycerate kinase for ATP in mesophyll chloroplast extracts of maize. (7) When the effect of adenylate energy charge on the state of activation and the direct effect on catalysis of the dikanase are combined, the total capacity for catalysis is very dependent on the energy charge.     

Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide. Protection by substrates and products

Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N-ethylmaleimide at micromolar concentrations. However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained. When incubated with 7.2 microM N-ethylmaleimide, acetate kinase was inactivated with a rate constant of 0.063 min-1. Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation. While both acetate and acetyl phosphate did not affect the protection rendered by AMP, the presence of acetyl phosphate altered ADP protection. However, both substrates prevented ATP from protecting the enzyme. These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides.