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.     

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.     

Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions.

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than Km for MgATP2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP leads to ATP + creatine) equal to 3.5 mumol times min-1 times mg-1 is essentially higher than maximal rate of the forward reaction (0.8 mumol times min-1 times mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reactions mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be expained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP AND ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.     

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

ATP and pyrophosphate at high concentration (> 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 magnesium-phosphate (Mg · P_i) complexes.It is highly probable that Mg · ADP^? and Mg · P_i, 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.     

Reaction mechanism of calcium-ATPase of sarcoplasmic reticulum. Substrates for phosphorylation reaction and back reaction, and further resolution of phosphorylated intermediates

Reaction of the purified Ca2+-ATPase of sarcoplasmic reticulum at 0 degrees C at low [gamma-32P]ATP (0.1 to 0.67 microM) and enzyme (0.025 to 0.24 microM) concentration in the presence of 0.11 to 30 mM Ca2+ without added Mg2+ has resulted in the formation of phosphorylated intermediate (EP:maximal level of EP = 0.45 mol/mol of enzyme) at a very slow rate. Under these conditions, the reaction steps in which EP decomposition takes place are completely prevented. This has permitted us to study the EP formation reaction and its reversal specifically, with a considerably improved time resolution. An apparent rate constant of EP formation (Vf) increases in parallel with the concentration of Ca . ATP, but not with those of Mg . ATP, or of protonated or fully ionized free ATP. This suggests that Ca . ATP is the substrate under these conditions. If Co2+ or Mn2+ are in excess over the other ions during the reaction, Vf varies in parallel with [Co . ATP] or [Mn . ATP]. Thus, it appears that either Ca2+, Co2+, or Mn2+ can be complexed with ATP to form the effective substrate. An apparent rate constant of the back reaction of EP initiated by addition of ADP to EP (Vr) increases in proportion to [ADP] or [H . ADP], but is inhibited by increasing concentrations of the ADP complex with Ca2+ or Mg2+, indicating that free ADP or protonated ADP, or both, are actual substrates for the back reaction of EP. These results suggest a new type of site to which the metal moiety of metal . ATP complex remains bound after the release of ADP from the enzyme. An acid-stable phosphorylated intermediate (EP) produced in the presence of high Ca2+ concentrations (e.g. 0.11 mM) without added Mg2+ does not decompose spontaneously, and the major portion (approximately 90%) of this EP (EPD+) reacts with ADP to form ATP (ADP-sensitive). Upon chelating Ca2+ with ethylene glycol bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), EPD+ is converted to another form of EP (EPD-), which is unreactive with ADP (or ADP-insensitive). Addition of Mg2+, after initiation of the reaction leading to EPD- by EGTA, results in rapid production of Pi from a portion of EPD- with KMg approximately equal to 3.3 x 10(3) M-1. The fraction of EPD- that is Mg2+-sensitive (EPD-,M+) increases with reaction time at a much slower rate than the Mg2+-insensitive portion of EPD- (EPD-,M-). These results suggest that the enzyme reaction involves the sequential formation of at least three forms of acid-stable EP, viz. in the order of formation, EPD+, EPD-,M-, and EPD-,M+. The equilibrium between EPD+ and EPD-,M- is shifted by higher [K+] and [Ca2+] towards EPD+.     

The alpha beta-methylene analogues of ADP and ATP act as substrates for creatine kinase. delta G0 for this reaction and for the hydrolysis of the alpha beta-methylene analogue of ATP

The alpha beta-methylene analogues of ATP and ADP, [alpha beta CH2]ATP and [alpha beta CH2]ADP, are substrates for creatine kinase. However, the rate of the phosphoryl transfer reaction catalysed is about 10(-5)-times lower than that with normal ATP. The affinities of the analogues (especially [alpha beta CH2]ADP) for the enzyme are lower than those of the normal substrates. The equilibrium constant at 25 degrees C, measured using 31P NMR, for the reaction Mg[alpha beta CH2]ATP + creatine in equilibrium Mg[alpha beta CH2]ADP + phosphocreatine + H+ is 2.2 X 10(-12) M compared with a value of 2.5 X 10(-10) M for the same reaction with the normal substrates, corresponding to a difference in delta G0 values of 11.7 kJ X mol-1. It follows that delta G0 for the hydrolysis of the terminal phosphate group of Mg[alpha beta CH2]ATP is less favourable by 11.7 kJ X mol-1 than that for MgATP.     

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.     

Influence of divalent cations on the reconstituted ADP, ATP exchange

1. Divalent cations cause a decrease in the exchange activity of the reconstituted ADP,ATP translocator from beef heart mitochondria. This effect is due to complex formation with the adenine nucleotides. 2. It is confirmed that only the free nucleotides are transported. A possible competition of free adenine nucleotides and the Mg2+-complexes for the binding site at the carrier protein is excluded. 3. The stability constants (Kn) for the cation-nucleotide complexes are derived from these experiments. For Mg2+-ATP, Kn = 0.8 x 10(4) M-1 and for Mg2+-ADP, Kn = 0.8 x 10(3) M-1 is obtained. 4. The carrier system was reconstituted with the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine. Interaction of the divalent cations with these phospholipids seem not to be important for the exchange suppression.     

Carbamate kinase of Lactobacillus buchneri NCDO110. II. Kinetic studies and reaction mechanism

The participation of Mg2+ or Mn2+ nucleoside diphosphates in the reverse reaction catalyzed by purified carbamate kinase (ATP:carbamate phosphotransferase, EC 2.7.2.2) of Lactobacillus buchneri NCDO110 was studied. The results of initial velocity studies have indicated that Mn2+ ADP is as effective as a substrate as Mg2+ ADP is. Product inhibition studies have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate and the other for carbamyl phosphate. The reaction of the enzyme with the substrates is of the random type.     

Ubiquitin adenylate: structure and role in ubiquitin activation

The acid precipitate of the ubiquitin activating enzyme after reaction with ATP and ubiquitin contains one enzyme equivalent of ubiquitin adenylate in which the carboxyl-terminal glycine of ubiquitin and AMP are in an acyl-phosphate linkage. The recovered ubiquitin adenylate has the catalytic properties proposed for it as a reaction intermediate. Thus, upon reaction with fresh enzyme in the absence of Mg2+ or ATP, the product complex, E-ubiquitin . AMP-ubiquitin, is formed. This complex is capable of generating ubiquitin-protein isopeptide derivatives when added to a reticulocyte fraction that catalyzes protein conjugation. This reproduces the effect previously shown to require ubiquitin, ATP, and Mg2+. In the presence of activating enzyme, ubiquitin adenylate is converted to ATP and free ubiquitin in a step requiring PPi and Mg2+. On the basis of studies of [32P]PPi/nucleoside triphosphate exchange, the activating enzyme could be used to generate 2'-deoxy-AMP-, 2'-deoxy-IMP-, and 2'-deoxy-GMP-ubiquitin but not pyrimidine nucleotide-ubiquitin derivatives. The enzyme shows a modest preference for the pro-S diastereomers of adenosine 5'-O-(1-thiotriphosphate) and adenosine 5'-O-(2-thiotriphosphate). Inorganic phosphate, arsenate, methyl phosphate, and tripolyphosphate, but not nucleoside triphosphates, can serve as alternate substrates in place of PPi in the reverse of ubiquitin adenylate formation. Therefore, the enzyme catalyzes the unusual reaction ATP + Pi in equilibrium ADP + PPi in the presence of ubiquitin.     

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [3H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [3H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [3H]ADP in 30 min with a Kd of 30 microM. [3H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [3H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [3H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits. It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.     

Regulation of steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells

Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed.     

Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; K(m) values are 4x10(-5)m for ATP and 6.3x10(-5)m for ADP. V(max.) for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg(2+) ions to form MgATP(2-) or MgADP(-) complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with K(m) values identical with those of the respective free nucleotides. 3. Mg(2+) ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive-non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg(2+) ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations.     

ATP-Mg/Pi carrier activity in rat liver mitochondria.

The ATP-Mg/Pi carrier in liver mitochondria is activated by micromolar Ca2+ and mediates net adenine nucleotide transport into and out of the mitochondrial matrix. The purpose of this study was to characterize certain features of ATP-Mg/Pi carrier activity that are essential for understanding how the mitochondrial adenine nucleotide content is regulated. The relative importance of ATP and ADP as transport substrates was investigated using specific trap assays to measure their separate rates of carrier-mediated efflux with Pi as the external counterion. Under energized conditions ATP efflux accounted for 88% of total ATP+ADP efflux. With oligomycin present to lower the matrix ATP/ADP ratio, ATP efflux was eliminated and ADP efflux was relatively unaffected. Mg2+ was stoichiometrically required for ATP influx and is probably transported simultaneously with ATP. Ca2+ and Mn2+ could substitute for the stoichiometric Mg2+ requirement. ADP influx and Pi-induced adenine nucleotide efflux were unaffected by external Mg2+. Experiments with Pi analogues suggested that Pi is transported as the divalent anion, HPO4(2-). The results show that ATP-Mg and divalent Pi are the major transport substrates; the most probable transport mechanism for the ATP-Mg/Pi carrier is an electroneutral exchange. The results are consistent with the hypothesis that the direction and magnitude of net adenine nucleotide movements are determined mainly by the (ATP-Mg)2- and HPO4(2-) concentration gradients across the inner mitochondrial membrane.     

Structure of the metal-nucleotide complex in the acetate kinase reaction. A study with gamma-32P-labeled phosphorothioate analogs of ATP

The synthesis of the gamma-32P-labeled diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and the Sp isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) by a modification of the Glynn and Chappell method (Glynn, I. M., and Chappell, J. T., (1964) Biochem. J. 90, 147-149) is described. These analogs were tested as substrates for acetate kinase in the presence of several divalent metal ions. Both isomers of ATP alpha S are substrates in the presence of Mg2+, Mn2+, Co2+, Zn2+, and Cd2+, the Sp isomer being preferred by a factor of between 4.8 (Mg2+) and 52.5 (Cd2+). Only the Rp isomer of ATP beta S is a substrate in the presence of Mg2+, and the Sp isomer becomes a better substrate in the presence of Mn2+, Co2+, and Zn2+; both isomers are equally good substrates in the presence of Cd2+. The change in specificity upon replacing Mg2+ by Cd2+ is greater than 1800 at beta-phosphorus and 10 at alpha phosphorus. These results provide a basis for proposing that the lambda screw sense configuration of the beta, gamma-bidentate MgATP complex is the substrate for acetate kinase. In the reverse reaction, both Sp and Rp isomers of ADP alpha S are substrates in the presence of all metal ions tested, the Sp isomer preferred by a factor between 12.3 (Mg2+) and 45.5 (Cd2+). In the presence of Mg2+, Mn2+, and Co2+, only the Rp isomer of ATP beta S is synthesized from prochiral ADP beta S, while a mixture of Rp and Sp isomers is synthesized in the presence of Zn2+ and Cd2+. These results are analogous to those for the forward reaction and suggest that the Mg.ADP complex which binds as a substrate in the reverse reaction, and is released as a product in the forward reaction, is the beta-monodentate. The classification of acetate kinase as an enzyme having a type I mechanism (Dunaway-Mariano, D. and Cleland, W. W. (1980) Biochemistry 19, 1506-1515) for kinases, is discussed.     

The interaction of phosphorothioate analogues of ATP with phosphomevalonate kinase. Kinetic and 31P NMR studies

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma-bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor.     

Matrix free Mg2+ changes with metabolic state in isolated heart mitochondria

The concentration of free Mg2+ in the matrix of isolated heart mitochondria has been monitored by using the fluorescent probe furaptra (mag-fura-2). Beef heart mitochondria respiring in a KCl medium in the absence of external Mg2+ maintain free matrix Mg2+ near 0.50 mM. Addition of Pi under these conditions decreases free Mg2+ by 0.12-0.17 mM depending on the substrate. This decrease in free Mg2+ appears to reflect changing ligand availability in the matrix. The decrease is prevented when the Pi transporter is blocked by mersalyl. Addition of ADP to initiate state 3 respiration causes a marked increase in free matrix Mg2+ (0.1-0.2 mM) that persists as long as ATP formation is taking place; free Mg2+ then returns to the base level. This cyclic change is blocked by oligomycin and carboxyatractyloside and appears to reflect to a large extent the decrease in matrix Pi that accompanies oxidative phosphorylation. Exchange of external ADP for matrix ATP may also contribute to the increase in free matrix Mg2+. Addition of an uncoupler promotes anion efflux and increases free matrix Mg2+. Similar changes in free Mg2+ on addition of Pi, ADP, or uncoupler are seen when extramitochondrial Mg2+ is buffered from 0.5 to 2 mM, but the basal free matrix Mg2+ increases as external Mg2+ concentration increases in this range. Free matrix Mg2+ also increases when total mitochondrial Mg2+ is increased by respiration-dependent uptake in the presence of Pi. It is concluded that matrix free Mg2+ changes significantly with changing ligand availability and that such changes may contribute to the regulation of Mg2(+)-sensitive matrix enzymes and membrane transporters.     

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.     

Kinetic studies on the ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase. Evidence for the K.S.T. mechanism with two enzyme-ATP complexes and two phosphorylated intermediates of high-energy type.

The kinetic properties of the [3H]ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase [EC 3.6.1,3] were investigated, using NaI-treated microsomes from bovine brain, and the following results were obtained. 1. The rates of the Na+-dependent exchange reaction in the steady state were measured in a solution containing 45 micronM free Mg2+, 100 mMNaCl, 80 micronM ATP, and 160 micronM ADP at pH 6.5 and 4-5 degrees. The rate and amount of decrease in phosphorylated intermediate on adding ADP, i.e., the amount of ADP-sensitive EP, were measured while varying one of the reaction parameters and fixing the others mentioned above. Plots of the exchange rate and the amount of ADP-sensitive EP against the logarithm of free Mg2+ concentration gave bell-shaped curves with maximum values at 50-60 micronM free Mg2+. Plots of the exchange rate and the amount of ADP-sensitive EP against pH also gave bell-shaped curves with maximum values at pH 6.9-7. They both increased with increase in the concentration of NaCl to maximum values at 150-200 mM NaCl, and then decreased rapidly with increase in the NaCl concentration above 200 mM. The dependences of the exchange rate and the amount of ADP-sensitive EP on the concentration of ADP followed the Michaelis-Menten equation, and the Michaelis constants Km, for both were 43 micronM. The dependence of the exchange rate on the ATP concentration also followed the Michaelis-Menten equation, and the Km value was 30 micronM. The amount of ADP-sensitive EP increased with increase in the ATP concentration, and reached a maximum value at about 5 micronM ATP. 2. The N+-dependent [3H]ADP-ATP exchange reaction was started by adding [3H]ADP to EP at low Mg2+-concentration. The reaction consisted of a rapid initial phase and a slow steady phase. The amount of [3H]ATP formed during the rapid initial phase, i.e. the size of the ATP burst, was equal to that of ADP-sensitive EP, and was proportional to the rate in the steady state. At high Mg2+ concentration, the rate of Na+-dependent exchange in the steady state was almost zero, and EP did not show any ADP sensitivity. However, rapid formation of [3H]ATP was observed in the pre-steady state, and the size of the ATP burst increased with increase in the KCl concentration. From these findings, we concluded that an enzyme-ATP complex (E2ATP) formed at low Mg2+ concentration is in equilibrium with EP + ADP, that the rate-limiting step for the exchange reaction is the release of ATP from the enzyme-ATP complex, that the ADP-insensitive EP (formula: see text) produced at high Mg2+ concentration is in equilibrium with the enzyme-ATP complex, and that the equilibrium shifts towards the enzyme-ATP complex on adding KCl. Actually, the ratio of the size of the ATP burst to the amount of EP was equal to the reciprocal of the equilibrium constant of step (formula: see text), determined by a method previously reported by us.     

Effects of magnesium and ATP on pre-steady-state phosphorylation kinetics of the Na+,K(+)-ATPase.

The aim of the present work was to elucidate the role played by ATP and Mg2+ ions in the early steps of the Na+,K(+)-ATPase cycle. The approach was to follow pre-steady-state phosphorylation kinetics in Na(+)-containing K(+)-free solutions under variable ATP and MgCl2 concentrations. The experiments were performed with a rapid mixing apparatus at 20 +/- 2 degrees C. The concentrations of free and complexes species of Mg2+ and ATP were calculated on the basis of a dissociation constant of 0.091 +/- 0.004 mM, estimated with Arsenazo III under identical conditions. A simplified scheme were ATP binds to the ENa enzyme, which is phosphorylated to MgEPNa and consequently dephosphorylated returning to the ENa form, was used. In the absence of ADP and phosphate four rate constants are relevant: k1 and k-1, the on and off rate constants for ATP binding; k2, the transphosphorylation rate constant and k3, the constant that governs the dephosphorylation rate. The values obtained were: k1 = 0.025 +/- 0.003 microM-1 ms-1 for both free ATP and ATPMg; k-1 = 0.038 +/- 0.004 ms-1 for free ATP and 0.009 +/- 0.002 ms-1 for ATPMg; k2 = 0.199 +/- 0.005 ms-1; k3 = 0.0019 +/- 0.0002 ms-1. The model that seems best to explain the data is one where (i) the role of true substrate can be played equally well by free ATP or ATPMg, and (ii) free Mg2+, an essential activator, acts by binding to a specific Mg2+ site on the enzyme molecule.