Bovine lenticular gamma-glutamylcysteine synthetase: reaction sequence.

The sequence of substrate addition and product release during the reaction catalyzed by gamma-glutamylcysteine synthetase was investigated with purified enzyme from bovine lens. Thermal inactivation and kinetic studies suggest that L-glutamate is the first substrate to bind to the enzyme. L-beta-Chloroalanine was used as the L-cysteine analogue. Utilizing substrate activation and product inhibition studies, the following reaction sequence was determined: L-glutamate binding. ATP binding, ADP release, L-beta-chloroalanine binding, followed by inorganic phosphate and then dipeptide release. The implications of this mechanism with regard to control of the enzyme in situ and its importance in glutathione synthesis are discussed.     

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.     

基因工程酶法生产谷胱甘肽的工艺研究

研究了前体氨基酸和辅因子对基因工程酶法生产谷胱甘肽(GSH)的影响.实验考察了不同浓度的前体氨基酸和不同的镁离子浓度对于重组大肠杆菌酶法生产GSH产量的变化.实验结果表明,当L-Glu 60 mmol·L-1;Gly 50 mmol·L-1;L-Cys 15 mol·L-1;Mg2+ 80 mmol·L-1为酶法生产谷...     

ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm)

ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.     

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.     

Kinetic properties of type-II ATP diphosphohydrolase from the tunica media of the bovine aorta.

The kinetic properties of type-II ATP diphosphohydrolase are described in this work. The enzyme preparation from the inner layer of the bovine aorta, mostly composed of smooth muscle cells, shows an optimum at pH 7.5. It catalyzes the hydrolysis of tri- and diphosphonucleosides and it requires either Ca2+ or Mg2+ for activity. It is insensitive to ouabain (3 mM), an inhibitor of Na+/K(+)-ATPase, to tetramisole (5 mM), an inhibitor of alkaline phosphatase, and to Ap5A (100 microM), an inhibitor of adenylate kinase. In contrast, sodium azide (10 mM), a known inhibitor for ATPDases and mitochondrial ATPase, is an effective inhibitor. Mercuric chloride (10 microM) and 5'-p-fluorosulfonylbenzoyl adenosine are also powerful inhibitors, both with ATP and ADP as substrates. The inhibition patterns are similar for ATP and DP, thereby, supporting the concept of a common catalytic site for these substrates. Apparent Km and Vmax, obtained with ATP as the substrate, were evaluated at 23 +/- 3 microM and 1.09 mumol Pi/min per mg protein, respectively. The kinetic properties of this enzyme and its localization as an ectoenzyme on bovine aorta smooth muscle cells suggest that it may play a major role in regulating the relative concentrations of extracellular nucleotides in blood vessels.     

Energy interconversion in sarcoplasmic reticulum vesicles in the presence of Ca2+ and Sr2+ gradients

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle are able to accumulate Ca2+ or Sr2+ at the expense of ATP hydrolysis. Depending on the conditions used, vesicles loaded with Ca2+ can catalyze either an ATP in equilibrium Pi exchange or the synthesis of ATP from ADP and Pi. Both reactions are impaired in vesicles loaded with Sr2+. The Sr2+ concentration required for half-maximal ATPase activity increases from 2 microM to 60-70 microM when the Mg2+ concentration is raised from 0.5 to 50 mM. The enzyme is phosphorylated by ATP in the presence of Sr2+. The steady state level of phosphoenzyme varies depending on both the Sr2+ and Mg2+ concentrations in the medium. Phosphorylation of the enzyme by Pi is inhibited by both Ca2+ and Sr2+. In the presence of 2 and 20 mM Mg2+, half-maximal inhibition is attained in the presence of 4 and 8 microM Ca2+ or in the presence of 0.24 mM and more than 2 mM Sr2+, respectively. After the addition of Sr2+, the phosphoenzyme is cleaved with two different rate constants, 0.5-1.5 s-1 and 10-18 s-1. The fraction of phosphoenzyme cleaved at a slow rate is smaller the higher the Sr2+ concentration in the medium. Ca2+ inhibition of enzyme phosphorylation by Pi is overcome by the addition of ITP. This is not observed when Ca2+ is replaced by Sr2+.     

Enzymatic characterisation of the multifunctional enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-alpha-aminoadipic-acid-dependent ATP/PPi exchange could be detected. Other L-cysteine- and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with alpha-aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid.     

Effects of organic acids on the synthesis of citrulline by intact rat liver mitochondria

Citrulline synthesis, mostly regulated at the carbamoyl-phosphate synthase I (EC 6.3.4.16) step by the intramitochondrial concentration of ATP and/or N-acetylglutamate is tested with four organic acids: propionate, alpha-ketobutyrate, dipropyl-acetate and 4-pentenoate. In the presence of 10 mM succinate, as the oxidizable substrate, citrullinogenesis was only inhibited by propionate and 4-pentenoate. With 10 mM L-glutamate, a significant inhibition was observed with the four acids. After the addition of ATP and N-acetylglutamate to uncoupled mitochondria, no inhibition could be demonstrated with dipropylacetate and 4-pentenoate. However, a slight inhibition remained with propionate and alpha-ketobutyrate. When mitochondria were incubated with 10 mM L-glutamate, ATP decreased with propionate, dipropylacetate and 4-pentenoate. Under the same conditions, N-acetylglutamate synthesis was strongly inhibited by each organic acid. The decrease of N-acetylglutamate synthesis was related to the constant diminution of intramitochondrial acetyl-coenzyme A (CoA) and to the increase of propionyl-CoA with propionate and alpha-ketobutyrate. Acetyl-CoA and propionyl-CoA are respectively substrate and competitive inhibitor of the N-acetylglutamate synthase (EC 2.3.1.1). Each acid displayed its optimum inhibition at concentrations between 1 and 2 mM. At these acid concentrations, mitochondria had the lowest acetyl-CoA content and the highest propionyl-CoA content.     

Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum.

A protein kinase which phosphorylated histone and protamine was partially purified from bovine cerebellum. Casein and phosvitin were inert as substrates. The enzyme did not require any cyclic nucleotide. A sulfhydryl compound such as 2-mercaptoethanol, glutathione, or cysteine was necessary for the reaction. The optimum pH was 8.5 to 9.0 Km values for ATP and whole histone were 3.3 X 10(-6) M and 150 microgram/ml, respectively. The optimum concentration of Mg2+ varied with histone fractions employed; with H2B histone as substrate the enzyme was most active at 50 to 100 nM Mg2", whereas with H1 and H2A histones the maximum activity was observed at 5 to 10 mM Mg2+ and with H3 and H4 histones the enzyme was active over a range of 5 to 75 mM Mg2+. The enzyme phosphorylated Ser-32 and Ser-36 in H2B histone and Ser-38 in H1 histone, although the reaction with Ser-36 in H2B histone was very slow. The molecular weight was 6.4 X 10(4). The sedimentation coefficient and Stokes radium were about 4.5 and 29 A, respectively. The enzyme showed heterogeneity upon isoelectrofocusing electrophoresis with isoelectric points of 5.6, 6.0, and 6.6. The enzyme was not inhibited by protein inhibitor nor by the regulatory subunit of cyclic AMP-dependent protein kinase. Preliminary analysis suggested that the enzyme was produced from its precursor protein by a limited proteolytic reaction.     

Regulation of the synthesis and hydrolysis of ATP by mitochondrial ATPase. Role of Mg2+

ATP hydrolysis, the exchange of inorganic phosphate with ATP, and ATP synthesis have been studied as a function of Mg2+ concentration in bovine heart submitochondrial particles. The rate of exchange is low at concentrations of Mg2+ below 3 mM, at higher concentrations, the exchange is several times higher. ATP hydrolysis shows a different pattern with respect to the concentration of Mg2+. The ratio of ATP hydrolyzed to ATP exchanged is above 20 at Mg2+ concentrations below 3 mM and about 5 at high Mg2+ concentrations; ADP induces a further drop of the ratio (2-3). By assays of the sensitivity of the hydrolytic reaction to organic solvents (dimethyl sulfoxide), it has been possible to determine the rate-limiting step of ATP hydrolysis. At 3 mM Mg2+, the rate-limiting step is the release of ADP in the soluble, but not in the particulate enzyme. However at higher Mg2+ concentrations, the rate-limiting step in the particulate enzyme is also ADP release. Therefore, the decrease in the ratio of ATP hydrolysis to inorganic phosphate incorporated into ATP coincides with a change in the kinetics of the enzyme, i.e. when the terminal step of ATP hydrolysis becomes rate-limiting, the inorganic phosphate-ATP exchange increases. Ca2+ induces an increase in the phosphate-ATP exchange at low Mg2+ concentrations.     

Effect of eosin Y on Ca2+ -independent, Mg2+ -dependent ATPase activity of smooth muscle cell plasma membranes

The effect of eosin Y (2',4',5',7'-tetrabromofluorescin) on basic kinetic parameters of the reaction of Mg2+ -dependent hydrolysis of ATP catalysed "basal" Mg2+ -ATPase myometrial cells plasma membrane has been studied. The eosin Y (10-100 microM) inhibited initial maximal velocity of the "basal" Mg2+ -ATPase of plasma membrane assayed for Mg2+ and ATP. At the same time the given inhibitor reduces the affinity of Mg2+ -ATPase for ATP. However, the difficult effect of the inhibitor action is observed for Mg ions: eosin Y in concentration of 10-50 microM increases the enzyme affinity for the ion-activator, while in concentration of 100 microM the affinity of Mg2+ -ATPase for Mg2+ is reduced. An analysis of eosin Y effect on catalytic efficiency of "basal" Mg2+ -ATPase of plasma membrane has shown, that at saturating concentrations of ATP (1 mM) the enzyme activity is less sensitive to the action of inhibitor. On this basis the conclusion is made that ATP in high concentrations can compete with eosin Y for active centre of Mg2+ -ATPase of smooth muscle cells plasma membrane.     

Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp.

1. The adenylate cyclase in Trypanosoma brucei is located in the plasma membrane. 2. A partial kinetic analysis of the properties of the enzyme revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4mM. 3. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. 4. At high concentrations, Mg2+ inhibited the enzyme. Furthermore, the enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. 5. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than one binding site for Mg2+ on the enzyme. 6. A study of the patterns of product inhibition revealed little or no effect of 3':5'-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). This result suggests that the substrate-binding domain on T. brucei adenylate cyclase interacts mainly with the triphosphate portion of the ATP molecule. 7. The enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline, adenosine, GTP and guanyl-5'-yl imidodiphosphate. 8. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was also inhibited by relatively high concentrations of Ca2+ (1 mM).     

Regulation and properties of glutamine synthetase purified from Bacillus cereus

The glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600,000, and its subunit molecular weight is 50,000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+. The highest activity was obtained when 20 mM Mg2+ and 0.5 mM Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation X ATP complex were 1.03, 0.34, and 0.40 mM (Mn2+: ATP = 1: 1); 14.0, 0.47, and 0.91 mM (Mg2+: ATP = 4: 1); and 9.09, 0.45, and 0.77 mM (Mg2+: Mn2+: ATP = 4: 0.2: 1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) mumoles per min per mg protein, respectively. Mg2+-dependent glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+ in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor(s) was detected in crude extracts from B. cereus.     

Detailed kinetics and regulation of mammalian NAD-linked isocitrate dehydrogenase

A mathematical model is presented to describe the catalytic mechanism of mammalian NAD-linked isocitrate dehydrogenase (NAD-IDH), a highly regulated enzyme in the tricarboxylic acid cycle, a crucial pathway in energy metabolism and biosynthesis. The mechanism accounts for allosteric regulation by magnesium-bound isocitrate and EGTA and calcium-bound ATP and ADP. The developed model is used to analyze kinetic data for the cardiac enzyme and to estimate kinetic parameter values. Since the kinetic mechanism is expressed in terms of chemical species (rather than biochemical reactants), the model explicitly accounts for the effects of biochemical state (ionic strength, pH, temperature, and metal cation concentration) on the kinetics. Because the substrate isocitrate competes with allosteric activators (ATP and ADP) and an inhibitor (EGTA) for metal ion cofactors (Ca(2+) and Mg(2+)), the observed kinetic relationships between reactants, activator and inhibitor concentrations, and catalytic flux are complex. Our analysis reveals that under physiological conditions, the ADP/ATP ratio plays a more significant role than Ca(2+) concentration in regulating the enzyme's activity. In addition, the enzyme is highly sensitive to Mg(2+) concentration in the physiological range, pointing to a potential regulatory role of [Mg(2+)] in mitochondrial energy metabolism.     

Purification and properties of glutamine synthetase from liver of Squalus acanthias

Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456-462]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg2+, MgATP, and L-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and L-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1-7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5-10% ethylene glycol. The apparent Km values for MgATP, L-glutamate, and ammonia (NH4+-NH3) are 0.7, 11.0, and 0.015 mM, respectively. Mg2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn2+ cannot replace Mg2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent Km for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine-N-oxide. Except for activation by halogen anions and the very low apparent Km for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent Km for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis.     

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Purification and catalytic properties.

5-Oxo-L-prolinase, an enzyme that catalyzes the conversion of 5-oxo-L-proline (L-pyroglutamate; L-2-pyrrolidone-5-carboxylate) to L-glutamate coupled with the cleavage of ATP to ADP and Pi, has been purified about 1600-fold from rat kidney. Purification was carried out in the presence of 5-oxo-L-proline which protects the enzyme under a variety of conditions. An estimate of the molecular weight (about 325,000) was made by gel filtration on Sephadex G-200. K+ (or NH4+) and Mg2+ were required for activity. GTP, ITP, CTP, and UTP were much less active than ATP; dATP was 43% as active as ATP. ADP inhibited and addition of pyruvate kinase and phosphoenolpyruvate activated the reaction. The enzyme, which is protected during storage by dithiothreitol, is inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and iodoacetamide. The apparent Km values for 5-oxo-L-proline and ATP are, respectively, 0.05 and 0.17 mM. The pH profile indicates a broad range of activity from about pH 5.5 to pH 11.2 with apparent maxima at about pH 7 and pH 9.7. The formation of Pi and glutamate was equimolar over a wide pH range. When the enzyme was incubated with ATP, Mg2+, K+, and L-2-imidazolidone-4-carboxylate or L-dihydroorotate, cleavage of ATP to ADP and Pi occurred, but no cleavage of the imino acid substrates was observed; when the enzyme was incubated under these conditions with 2-piperidone-6-carboxylate, 4-oxy-5-oxoproline, and 3-oxy-5-oxoproline, the corresponding dicarboxylic amino acids were formed, but the molar ratio of Pi to amino acid formation was significantly greater than unity.     

Steady-state kinetic properties of phosphoglycerate kinase of mung beans

Steady-state kinetics of the action of mung bean phosphoglycerate kinase have been investigated using 3-phosphoglycerate and ATP as substrates in the presence of Mg2+ ions. Keeping a constant and high Mg2+ concentration and varying the concentration of one of the substrates (ATP or 3-phosphoglycerates) at several fixed concentrations of the other substrate (3-phosphoglycerate or ATP), the Km values of Mg.ATP2- and 3-phosphoglycerate were found to be 0.42 and 0.60 mM, respectively. These values are independent of the concentration of the other substrate. A limiting value of Vmax, where the enzyme is saturated with both the substrates, was found to be 39.4 mumoles product formed per min per mg enzyme protein. This corresponds to a turnover number equal to 31.5 sec-1 (for molecular weight of the enzyme equal to 48,000). If [Mg2+] and [ATP4-] are held equal and varied together at several fixed concentrations of 3-phosphoglycerate, deviations from Michaelis-Menten kinetics (non-linear Lineweaver-Burk plots) are observed at lower values of ATP4- and Mg2+ (less than 0.1 mM), giving rise to apparent sigmoidicity in the rate versus [ATP4-] plots. It has been suggested that the real substrate for this enzyme is the Mg.ATP2- complex (and not free ATP4-). The complex dissociates at lower values of [Mg2+] and [ATP4-]. The resulting disproportionate decrease in the concentration of the complex brings about a steeper fall in the rate of reaction than is required by the Michaelis-Menten equation, giving rise to an apparent sigmoidicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Catalytic and regulatory properties.

Some kinetic properties of N-acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L-glutamate 5-phosphotransferase EC 2.7.2.8) purified approx. 2000-fold from Pseudomonas aeruginosa have been studied. The enzyme required Mg2+ for activity. Mn2+, Zn2+, Co2+, and Ca2+, in this order, could replace Mg2+ partially. The substrate specificity was narrow: N-carbamoyl-L-glutamate and N-formyl-L-glutamate were phosphorylated, but at a lower rate than N-acetyl-L-glutamate; N-propionyl-L-glutamate was almost inactive as a substrate. dATP, but neither GTP nor ITP, could be used instead of ATP. The enzyme had a broad pH optimum from pH 6.5 to 9. Feedback inhibition by L-arginine was markedly dependent on pH. Above pH 9 no inhibition was observed. L-Citrulline was three times less potent an inhibitor than L-arginine. The enzyme showed Michaelis-Menten kinetics, even at low concentration of the second substrate. The apparent Km was 2 mM for N-acetyl-L-glutamate (at 10 mM ATP) and approx. 3 mM for ATP (at 40 mM N-acetyl-L-glutamate). In the presence of L-arginine the rate-concentration curves for N-acetyl-L-glutamate became signoidal, while no cooperativity was detected for ATP. A method was developed allowing the determination of N-acetyl-L-glutamate in the nanomolar range by means of purified enzyme.     

Kinetic characteristics of glutamine synthetase from the chloroplasts of pea leaves

The kinetic properties of glutamine synthetase (EC 6.3.1.2) isolated from pea chloroplasts and purified according to the previously developed procedure have been investigated. The pH optimum for the enzymatic reaction in the presence of Mg2+ and Mn2+ are 7.5-7.6 and 5.5, respectively. The corresponding values of the activation energy per enzyme monomer (Mr = 60 000) are equal to 2900 and 1190 cal/mole. With Mg2+ the values of Km(app.) for NH4+, NH2OH, L-glutamate (+NH4+), L-glutamate (+NH2OH), ATP(+NH4+ and NH2OH) and Mg-ATP (+NH4+ and NH2OH) are 0.64, 17.5, 5.6, 7.0, 1.3 and 0.74 mM, respectively.