Kinetic studies on the reaction catalyzed by polynucleotide kinase from phage T4-infected Escherichia coli

Kinetic properties of polynucleotide kinase (EC 2.7.1.78) isolated from Escherichia coli cells infected with phage T₄ were investigated. The reaction depends on the concentration of MgATP, while free ATP or free Mg²⁺ have neither inhibitory nor accelerating effect. The initial reaction velocity was plotted against variable concentrations of ATP as the phosphate donor at various fixed concentrations of 5′-hydroxyl-DNA or oligo(rA) as the phosphate acceptor in the presence or absence of products. The double reciprocal plot analysis of the data suggested that the reaction obeys the random sequential mechanism. Various constants were determined and the reaction mechanism was discussed.     

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism

Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.     

Non-enzymatic ATP-mediated binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA

ATP mediates covalent binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA. This non-enzymatic reaction has been studied with 6-[14C]hydroxymethylbenzo[alpha]pyrene (]14C]BP-6-CH2OH) and 7-[14C]-hydroxymethylbenz[alpha]anthracene ([14C]BA-7-CH2OH) at 37 degrees C in Tris buffer (pH 7.0). While ADP mediates the reaction 25-50% as well as ATP, six other possible phosphate donors including AMP were inactive as cofactors. A complex response to ATP occurred in which low binding of BP-6-CH2OH or BA-7-CH2OH was observed at concentrations of ATP below 2.5 mM, but a greater than linear response to higher concentrations of ATP was observed until ATP was saturating. Binding of the substrates to RNA was much lower than to DNA. Fluorescence spectra of BP-6-CH2OH bound to DNA were almost identical to the spectra of 6-bromomethylbenzo[alpha]pyrene bound to DNA and free 6-methylbenzo]alpha]pyrene, indicating that ATP-mediated binding of BP-6-CH2OH to DNA occurs at the 6-methyl group. The fate of ATP and ADP in the binding reaction of BP-6-CH2OH was examined by thin layer chromatography. Loss of one phosphate group occurs during the reaction. With ATP the rate of loss is about 100-fold greater than the rate of binding of BP-6-CH2OH to DNA. This implies that the binding reaction proceeds through formation of a presumed reactive and unstable phosphate ester intermediate which then inefficiently binds to DNA.     

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

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

Estrogen-induced changes in high-energy phosphate metabolism in rat uterus: 31P NMR studies

Changes in the concentrations of high-energy phosphate metabolites were measured by 31P NMR spectroscopy of surviving rat uteri from 0-48 h following estrogen administration. Concentrations (millimoles per kilogram wet weight) of these metabolites in the untreated immature uterus, measured at 4 degrees C, were found to be the following: creatine phosphate (CP), 2.1 +/- 0.2; nucleoside triphosphates, mainly adenosine 5'-triphosphate (ATP), 4.6 +/- 0.4; phospho monoesters, primarily sugar phosphates (SP), 5.4 +/- 0.7; and inorganic phosphate (Pi), 0.8 +/- 0.4. Adenosine 5'-diphosphate (ADP) concentration was estimated to be approximately 40 mumol/kg wet weight from the assumed equilibrium of the creatine kinase reaction. The concentration of CP, and to lesser extent ATP and SP, declined within the first 1.5-3 h after injection of 17 beta-estradiol, returned to control values between 6 and 12 h, and then increased, reaching maximal concentrations at 24 h. From the fractions of the total soluble ATP in free and Mg2+-bound forms, [free Mg2+] in the untreated uterus was estimated to be 0.2-0.4 mmol/kg wet weight. An increase in [free Mg2+] in the uterus was detected 1.5 h after estrogen injection. A subsequent parallel increase in the ratio of ATP to CP concentrations suggests that estrogen can also affect the apparent creatine kinase equilibrium by modulating [free Mg2+].     

Kinetic mechanism and metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica

The kinetic mechanism and the metabolic role of pyruvate phosphate dikinase from Entamoeba histolytica were investigated. The initial velocity patterns in double reciprocal plots were parallel for the phosphoenolpyruvate/AMP and phosphoenolpyruvate/pyrophosphate substrate pairs and intersecting for the AMP/pyrophosphate pair. This suggests a kinetic mechanism with two independent reactions. The rate of ATP synthesis at saturating and equimolar concentrations of phosphoenolpyruvate, AMP, and pyrophosphate was inhibited by phosphate, which is consistent with an ordered steady-state mechanism. Enzyme phosphorylation by [(32)P(i)]pyrophosphate depends on the formation of a ternary complex between AMP, pyrophosphate, and pyruvate phosphate dikinase. In consequence, the reaction that involves the AMP/pyrophosphate pair follows a sequential steady-state mechanism. The product inhibition patterns of ATP and phosphate versus phosphoenolpyruvate were noncompetitive and uncompetitive, respectively, suggesting that these products were released in an ordered process (phosphate before ATP). The ordered release of phosphate and ATP and the noncompetitive inhibition patterns of pyruvate versus AMP and versus pyrophosphate also supported the sequential kinetic mechanism between AMP and pyrophosphate. Taken together, our data provide evidence for a uni uni bi bi pingpong mechanism for recombinant pyruvate phosphate dikinase from E. histolytica. The Delta G value for the reaction catalyzed by pyruvate phosphate dikinase (+2.7 kcal/mol) determined under near physiological conditions indicates that the synthesis of ATP is not thermodynamically favorable in trophozoites of E. histolytica.     

Properties and mechanism of action of pyruvate, phosphate dikinase from leaves

1. Sugar-cane leaf pyruvate,P(i) dikinase was prepared free of enzymes that would interfere with studies on the stoicheiometry and mechanism of the reaction it catalyses. The reaction was unequivocally shown to involve the conversion of equimolar amounts of pyruvate, ATP and P(i) into phosphoenolpyruvate, AMP and PP(i). 2. The purified enzyme was stable at pH8.3 only if stored at about 20 degrees in the presence of Mg(2+) and a thiol-reducing reagent, care being taken to prevent the oxidation of the thiol. 3. The apparent Michaelis constants for phosphoenolpyruvate and PP(i) were 0.11mm and 0.04mm respectively and that for AMP was less than 4mum. 4. At pH8.3 the initial velocity of the reaction was about 6 times as fast in the direction towards phosphoenolpyruvate synthesis as in the reverse direction. 5. With the exception of ATP, all the products of the reaction in both directions were inhibitory. 6. The phosphate groups of PP(i) were derived from P(i) and from the terminal phosphate of ATP. 7. Isotope-exchange studies indicated that the reaction proceeds in the following steps:Enzyme+ATP+P(i) right harpoon over left harpoon Enzyme-P+AMP+PP(i)Enzyme-P+pyruvate right harpoon over left harpoon Enzyme+phosphoenolpyruvate     

Endogenous phosphorylation of microsomal proteins in bovine corpus luteum. Tenfold activation by adenosine 3′:5′-cyclic monophosphate

Free ribosomes and a smooth-microsomal fraction were prepared from bovine corpus luteum. Both preparations will self-phosphorylate when incubated with Mg(2+) and ATP, but at low concentrations of Mg(2+) and ATP the self-phosphorylation of the smooth-microsomal fraction was much more dependent on cyclic AMP than was that of free ribosomes, stimulation by the nucleotide being up to 10-fold in the former case. The self-phosphorylation of the smooth-microsomal fraction was studied further. The reaction bears similarities to that brought about by soluble cyclic AMP-dependent protein kinase, being inhibited by Ca(2+) and the heat-stable inhibitor protein from skeletal muscle. Cyclic GMP will activate the reaction at concentrations higher than those required for full activation by cyclic AMP. In the presence of cyclic AMP, phosphate bound to protein is found almost exclusively as phosphoserine. Several proteins are phosphorylated, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the phosphorylation of all of them is markedly stimulated by cyclic AMP. If the reaction is carried out at high concentrations of Mg(2+) and ATP, a distinct cyclic AMP-independent phosphorylation is observed. This activity is not inhibited by the heat-stable inhibitor protein, and phosphate is found esterified with both threonine and serine residues.     

Energetics and the design principles of the Na/K-ATPase

Following on the pioneering analysis by Pickart and Jencks of the energetics of the calcium pump, the present mini review attempts a similar analysis of the somewhat more complicated sodium--and potassium--activated ATPase pump-enzyme. The analysis is based on the measurements of the rate-constants of the individual steps in the enzymic reaction which brings about pumping and uses data assembled by Stürmer et al., in the laboratory of Peter L?uger in Konstanz. The aim of such an analysis is to calculate the overall free energy released on ATP hydrolysis and to apportion this energy among the successive steps of the pump-enzyme reaction. We calculate the so-called basic free energy changes that take into account the prevailing ligand and ion concentrations, rather than the standard-state free energies that refer to 1 M concentrations of these ions and ligands. Using appropriate values of the ion and ligand concentrations in cardiac muscle, the available free energy which can be released from the hydrolysis of ATP (at 20 degrees C) comes out at 575 mV. Following a complete cycle of pumping, 371 mV of this free energy are found stored in the sodium and potassium ion gradients. The remaining 204 mV from the free energy of hydrolysis of ATP are lost to the ATP system. This part of the energy, that had been transduced into the concentration gradient of sodium, has presumably been used in the living cell to drive the co- and counter-transport (symport and antiport) of ions and metabolites in secondary transports. The free energy changes are pretty evenly apportioned along the various steps in the pumping cycle. The steps that one might naively have thought to be "powered", such as the step in which covalently bound phosphate is transformed from a high-energy to a low-energy state, or the step in which sodium is released into the phase containing a high concentration of sodium, show some of the lowest drops of free energy, 61 mV and 27 mV, respectively. The most surprising step in the overall reaction of ATP hydrolysis and synthesis is the phosphorylation of the protein from inorganic phosphate with formation of the acylphosphate bond. The stabilization of the acylphosphate bond presumably arises from ionic interactions between the covalently bound phosphate itself and appropriate groupings on the enzyme. ATP formation on the F0F1 ATPase (the F-type ATPase) is in an analogous way stabilized in the first place by phospho-ligand/enzyme interactions.     

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.     

32P]ATP synthesis in steady state from [32P]Pi and ADP by Na+/K(+)-ATPase from ox brain and pig kidney. Activation by K+

The ouabain-sensitive synthesis of [32P]ATP from [32P]Pi and ADP (vsyn) was measured in parallel with the ouabain-sensitive hydrolysis of [32P]ATP (vhy) at steady state, at varying concentrations of sodium, potassium, magnesium, inorganic phosphate, ADP, ATP and oligomycin, and at varying pH. Na+ was necessary for ATP synthesis, but vsyn was decreased by high sodium concentrations. Oligomycin, depending on the Na+ concentration, either decreased or did not affect vsyn. Potassium, at low concentrations (1-5 mM) increased vsyn at all magnesium and sodium concentrations tested, lower potassium concentrations being needed to activate vsyn at lower sodium concentrations. vsyn was optimal below pH 6.7, decreasing abruptly at higher values of pH. At pH 6.7, vsyn was a hyperbolic function of the concentration of inorganic phosphate. In the presence of potassium, half-maximal rate was obtained at [Pi] congruent to 40 mM, whereas a higher concentration was needed to obtain half-maximal rate in the absence of K+. In contrast, increasing the concentration of ADP caused a nonhyperbolic activation of vsyn, the pattern obtained in the presence of potassium being different from that obtained in its absence. Increasing the ATP concentration above 0.5 mM decreased vsyn. The data are used to elucidate (1) which reaction steps are involved in the ATP-synthesis catalysed by the Na+/K(+)-ATPase at steady state in the absence of ionic gradients and (2) the mechanism by which K+ ions stimulate the reaction.     

Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.     

Inhibition of photophosphorylation by ATP and the role of magnesium in photophosphorylation.

ATP and pyrophosphate at high concentration (greater than 1 mM) inhibited photophosphorylation of isolated spinach chloroplasts in the normal salt medium and did not cause stimulation of electron transport. The inhibition of photophosphorylation by ATP or pyrophosphate was shown to be abolished by the addition of excess MgCl2, ADP and phosphate. It has been demonstrated that the rates of photophosphorylation in the absence and presence of ATP or pyrophosphate are determined similarly by the concentrations of magnesium-ADP (Mg - ADP-) and magnesiumphosphate (Mg - Pi) complexes. It is highly probable that Mg - ADP- and Mg - Pi, but not free ADP and free phosphate, are the active form of the substrates of photophosphorylation. This is in support of the view that ATP inhibits photophosphorylation by decreasing the concentration of Mg2+ which is available for the formation of the complex with ADP and phosphate.     

The control of rat-heart phosphofructokinase by citrate and other regulators

1. The effects of ATP, inorganic phosphate and citrate on the relationship between fructose 6-phosphate concentration and initial velocity of reaction has been investigated with a partially purified preparation of rat-heart phosphofructokinase. 2. At low concentrations of ATP (<80mum) rate curves for fructose 6-phosphate approximated to Michaelis-Menten kinetics. At higher ATP concentrations rate curves were sigmoid, the K(m) for fructose 6-phosphate increased and the reaction appeared to be first-order with respect to fructose 6-phosphate at concentrations above its K(m) and of a higher order at concentrations below its K(m). Inorganic phosphate lowered the K(m) for fructose 6-phosphate and the concentration at which the apparent kinetic order decreased. 3. At 40mum-ATP, citrate was an activator at low concentration (<100mum) and an inhibitor at higher concentrations. At 0.5mm-ATP, citrate was inhibitory at all concentrations tested. 4. A new method for phosphofructokinase assay using [U-(14)C]fructose 6-phosphate is described which allows measurements to be made of the velocity of the forward reaction at known concentrations of the products of the reaction. With this method confirmatory evidence has been obtained that concentrations of ATP, AMP, phosphate and citrate may regulate phosphofructokinase in the perfused rat heart.     

Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase

To examine the role of changes in the distribution of the creatine kinase (CK) isoenzymes [BB, MB, MM, and mitochondrial CK (mito-CK)] on the creatine kinase reaction velocity in the intact heart, we measured the creatine kinase reaction velocity and substrate concentrations in hearts from neonatal rabbits at different stages of development. Between 3 and 18 days postpartum, total creatine kinase activity did not change, but the isoenzyme distribution and total creatine content changed. Hearts containing 0, 4, or 9% mito-CK activity were studied at three levels of cardiac performance: KCl arrest and Langendorff and isovolumic beating. The creatine kinase reaction velocity in the direction of MgATP production was measured with 31P magnetization transfer under steady-state conditions. Substrate concentrations were measured with 31P NMR (ATP and creatine phosphate) and conventional biochemical analysis (creatine) or estimated (ADP) by assuming creatine kinase equilibrium. The rate of ATP synthesis by oxidative phosphorylation was estimated with oxygen consumption measurements. These results define three relationships. First, the creatine kinase reaction velocity increased as mito-CK activity increased, suggesting that isoenzyme localization can alter reaction velocity. Second, the reaction velocity increased as the rate of ATP synthesis increased. Third, as predicted by the rate equation, reaction velocity increased with the 3-fold increase in creatine and creatine phosphate contents that occurred during development.     

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.     

Mutational analysis of carbamyl phosphate synthetase. Substitution of Glu841 leads to loss of functional coupling between the two catalytic domains of the synthetase subunit.

The synthetase subunit of Escherichia coli carbamyl phosphate synthetase has two catalytic nucleotide-binding domains, one involved in the activation of HCO3- and the second in phosphorylation of carbamate. Here we show that a Glu841----Lys841 substitution in a putative ATP-binding domain located in the carboxyl half of the synthetase abolishes overall synthesis of carbamyl phosphate with either glutamine or NH3 as the nitrogen source. Measurements of partial activities indicate that while HCO3(-)-dependent ATP hydrolysis at saturating concentrations of substrate proceeds at higher than normal rates, ATP synthesis from ADP and carbamyl phosphate is nearly completely suppressed by the mutation. These results indicate Glu841 to be an essential residue for the phosphorylation of carbamate in the terminal step of the catalytic mechanism. The Lys841 substitution also affects the kinetic properties of the HCO3- activation site. Both kcat and Km for ATP increase 10-fold, while Km for HCO3- is increased 100-fold. Significantly, NH3 decreases rather than stimulates Pi release from ATP in the HCO3(-)-dependent ATPase reaction. The increase in kcat of the HCO3(-)-dependent ATPase reaction, and an impaired ability of the Lys841 enzyme to catalyze the reaction of NH3 with carboxy phosphate, strongly argues for interactions between the two catalytic ATP sites that couple the formation of enzyme-bound carbamate with its phosphorylation.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the Pi-ATP exchange and hydrolytic reactions in F0-F1 reconstituted liposomes

The regulation of ATP hydrolysis and Pi-ATP exchange reactions by ATP, ADP, Mg2+, and phosphate was studied in liposomes containing F0-F1 obtained from bovine heart submitochondrial particles by solubilization with lauryl dimethylamino oxide as described previously (Dreyfus, G., Celis, H., and Ramirez, J. (1984) Anal. Biochem. 142, 215-220). A simultaneous analysis of ATP hydrolysis and the Pi-ATP exchange reactions showed that the ratio of hydrolysis/exchange is close to one when the ATP concentration is in the lower micromolar range. In this preparation ADP stimulates the Pi-ATP exchange reaction and depresses ATP hydrolysis. The exchange reaction is almost abolished when ADP is removed from the medium by an ATP-regenerating system. Mg2+ in millimolar concentrations stimulates Pi-ATP exchange, and at the same time decreases ATP hydrolysis; accordingly, the hydrolysis/exchange ratio depends on the concentration of Mg2+. Inorganic phosphate also controls this ratio, a lower ratio being observed at high phosphate concentrations. The Pi-ATP exchange reaction, but not ATP hydrolysis, depends on the concentration of medium phosphate. These results indicate that the kinetic characteristics of this F0-F1 preparation are modified by Mg2+, ATP, and phosphate.