Characterization of phosphate oxygen exchange reactions catalyzed by myosin through measurement of the distribution of 18-O-labeled species

The change in the distribution of the phosphate species containing 0 to 4 18O oxygens per Pi was investigated during medium Pi equilibrium HOH exchange catalyzed by myosin subfragment 1. At 25 degrees C, a Pi molecule once bound loses an average of 3.9 of its original 4 oxygens prior to release which means that at least 100 reversals of the exchange reaction must have occurred. At 0 degrees C, only 3.4 of the 4 oxygens are lost prior to release indicating an average of 17 reversals. Distribution patterns are consistent with equivalent participation in the exchange reactions of all 4 oxygens of bound Pi. The intermediate exchange of Pi oxygens during hydrolysis of 18O-labeled ATP by myosin has also been investigated. The distribution of the product Pi species shows that there is an ATPase component in myosin preparations which hydrolyzes ATP without intermediate exchange. Presence of this component, which is likely a contaminating ATPase, provides a simple explanation of the apparent nonequivalence of phosphate oxygens which has been observed. When correction is made for this contaminant, characteristics of the myosin intermediate Pi equilibrium HOH exchange are similar to those of myosin subfragment 1 medium exchange, and intermediate exchange data are in much closer agreement with other kinetic measurements.     

Evidence for energy-dependent change in phosphate binding for mitochondrial oxidative phosphorylation based on measurements of medium and intermediate phosphate-water exchanges.

Characteristics of the exchange reactions catalyzed by beef heart submitochondrial particles give new insight into energy transducing steps of oxidative phosphorylation. The uncoupler-insensitive portion of the total Pi in equilibrium HOH exchange in presence of ATP, ADP, and Pi is the intermediate Pi in equilibrium HOH exchange, that is the exchange occurring with Pi formed by hydrolysis of ATP prior to release of Pi from the catalytic site. The exchange of medium Pi with HOH is as sensitive to uncouplers as the Pi in equilibrium ATP exchange and net oxidative phosphorylation, demonstrating a requirement of an uncoupler-sensitive energized state, probably a transmembrane potential or proton gradient, for bringing medium Pi to the reactive state. The covalent bond forming and breaking step at the catalytic site (ADP + Pi in equilibrium ATP + HOH) appears relatively insensitive to uncouplers. Thus to the extent that uncouplers dissipate transmembrane proton-motive force, it is unlikely that such a force is used to drive ATP formation by direct protonations of Pi oxygens. When only Pi and ADP are added and formation of ATP from added ADP by adenylate kinase and subsequent ATP hydrolysis are adequately blocked, no Pi in equilibrium HOH exchange can be observed, demonstrating a requirement of energization by ATP binding and cleavage for such an exchange. This uncoupler-insensitive energization is suggested to represent a conformationally energized state that can be used reversibly to develop a transmembrane protonmotive force accompanying ADP and Pi release. Rates of various exchanges as estimated by improved procedures are compatible with all oxygen exchanges occurring by dynamic reversal of ATP hydrolysis at the catalytic site.     

The rapid labeling of adenosine triphosphate by 32P-labeled inorganic phosphate and the exchange of phosphate oxygens as related to conformational coupling in oxidative phosphorylation.

Evidence is presented that extends and amplifies the concept that in oxidative phosphorylation energy input serves to bring about release of ATP formed at a catalytic site by reversal of hydrolysis. The evidence with beef heart submitochondrial particles includes additional demonstration of uncoupler insensitive Pi leads to HOH exhchange, demonstration that this exchange is sensitive to the specific phosphorylation inhibitor, oligomycin, and demonstration that the small burst of uncoupler-insensitive ATP, rapidly labeled after addition of a tracer of 32Pi, behaves in a manner consistent with its participation as a membrane-bound intermediate in the Pi leads to HOH exchange. In addition, data are presented showing that addition of hexokinase plus glucose to submitochondrial particles in presence of ADP and Pi considerably lowers the Pi leads to HOH exchange but that further addition of cyanide or 2,4-dinitrophenol or both has little additional effect. Such data are compatible with no energy requirement for formation of bound ATP. However, with a large excess of hexokinase, the rate of the Pi leads to HOH exchange is further depressed. This could reflect some use of energy to promote formation of ATP at the catalytic site or to maintain the integrity of the phosphorylation system. Relationships of these findings to related information in the field are discussed.     

Induction by nucleotide triphosphate hydrolysis of a form of sarcoplasmic reticulum ATPase capable of medium phosphate-oxygen exchange in presence of calcium.

Sarcoplasmic reticulum vesicles rendered leaky by exposure to alkaline pH, like intact vesicles, catalyze a rapid Mg2+-dependent exchange of oxygens of medium Pi with water. The exchange with 10 mM Pi is strongly inhibited by 0.15 mM Ca2+. Upon addition and hydrolysis of ITP or ATP, a rapid phosphate-oxygen exchange is observed even with 0.15 mM Ca2+ present and a definite but smaller exchange at 8 mM Ca2+. Oxygen exchange per Pi formed is greater with ITP than with ATP. When no Pi is initially present, the extent of oxygen exchange is increased with time of incubation as Pi is formed. With 18O-labeled Pi present, ATP hydrolysis accelerates 18O loss. The results show that much of the oxygen exchange occurs as a result of reversible binding of medium Pi. Thus the binding and cleavage of ITP or ATP overcomes the Ca2+ inhibition of the medium Pi in equilibrium HOH exchange. Such findings support the concept that the cleavage cycle includes a transient conformational form which can reversibly react with Pi to give a phosphoryl enzyme and resultant oxygen exchange or in a rate-limiting step decay to a form with high Ca2+ and NTP affinity.     

Coupling of "high-energy" phosphate bonds to energy transductions.

Recent results suggest consideration of a new concept for oxidative phosphorylation in which a prime function of energy is to bring about release of ATP formed at the catalytic site by reversal of hydrolysis. Data with submitochondrial particles include properties of an uncoupler insensitive Pi=HOH exchange, a rapid reversible formation of bound ATP in presence of uncouplers, and predictable patterns of 32-Pi incorporation into ATP in rapid mixing experiments. ADP is confirmed as the primary Pi acceptor in mitochondrial ATP synthesis, but with chloroplasts ADP is also rapidly labeled. Other findings with pyrophosphatase and with transport ATPase harmonize with the new concept. Measurements of the reversal of ATP cleavage and binding by myosin suggest that oxygen exchanges result from reversible cleavage of ATP to ADP and Pi at the catalytic site and that the principal free energy change in ATP cleavage occurs in ATP binding. Reversal of conformational changes accompanying ATP binding and cleavage is proposed to drive the actin filament in contraction. Thus energy transductions linked to ATP in both mitochondria and muscle may occur primarily through protein conformational change.     

Two types of kinetic regulation of the activated ATPase in the chloroplast photophosphorylation system

The inhibition by light of chloroplast coupling factor ATPase is not due simply to competing photophosphorylation. This inhibition is only partially relieved by either an arsenate-pool trap for released phosphate, or a pyruvate kinase/phosphoenolpyruvate trap for ADP. Moreover, the amount of product return that does occur in the absence of trapping systems, ascertained by incorporation of 32Pi or [2-3H]ADP back into ATP during the hydrolysis reaction, is insufficient to account for the observed activity decrease. In intermediate pi:H2O oxygen exchange studies, the number of water oxygens incorporated into each molecule of Pi produced does not vary with light intensity during the ATPase assay. This indicates that the light-induced change in ATPase activity is not due to an alteration of rat constants involved in the forward and reverse partitioning of the E.ADP.Pi complex. In contrast, ammonium chloride, an uncoupler of photophosphorylation which stimulates membrane-bound coupling factor ATPase when added after light activation, causes a shift in the pattern of intermediate Pi:H2O oxygen exchange toward a lower number of water oxygens incorporated per Pi formed. The effect of NH4+ consistent with ATPase activity stimulation caused by enhanced partitioning forward of the E.products complex. These observations suggest the operation of two mechanisms of regulation of ATP ase activity during chloroplast de-energization. However, a direct effect of NH4+ on the coupling factor itself, independent of the membrane energization effect, cannot be ruled out by the present studies. Additional oxygen exchange experiments lead to the conclusion that the binding of ATP at a site catalyzing extensive ATP:H2O back exchange in the native chloroplast system ( Wimmer, M. J., and Rose, I. A. (1977) J. Biol. Chem. 252, 6769-6775) is different from the binding of ATP for net hydrolysis in the system activated for ATPase.     

Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules

The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.     

Deficiency of uncoupler-stimulated adenosine triphosphatase activity in yeast mitochondria

Oligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was dependent upon phosphate (Pi) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions, external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi-ATP exchange experiments. These results are discussed in terms of a limitation of the translocase-mediated ATP/ADP exchange in uncoupled mitochondria.     

Occurrence of an uncoupler-resistant intermediate type of phosphate-water oxygen exchange reaction catalyzed by heart submitochondrial particles

The hydrolysis of ATP catalyzed by phosphorylating vesicles prepared from bovine heart mitochondria by ultrasonic disruption was studied in H218O. Provided that an ATP-generating system was included to prevent accumulation of ADP due to hydrolysis, the addition of 20 mM arsenate or 0.5 mM 2,4-dinitrophenol to the incubation mixture either singly or together, had little or no effect on the number of oxygen atoms from H2O incorporated (on the average) into each molecule of Pi formed by hydrolysis (the O:P ratio). As the ATP concentration was reduced from 2.0 to 0.05 mM, the O:P ratio increased from about 1.4 to over 2.0 and, although dinitrophenol significantly increased the ATPase activity, it did not significantly alter the O:P ratio for a given ATP level. This implies that the uncoupler does not act directly on the terminal transphosphorylation step. Companion experiments were performed in which 18O label was placed either initially in H2O or Pi. Under conditions where extensive exchange from H218O into Pi occurred, no 18O was lost from medium Pi under identical circumstances, thus showing that the exchange was intermediate and did not involve medium Pi. Kinetic plots of v vs. v/S were nonlinear with respect to ATPase activity. The kinetic data, as well as the Pi = H218O exchange data, are consistent with enzyme models having multiple forms of catalytic sites. Several models are evaluated and attempts are made to distinguish between some of the simpler cases of these models.     

ATP hydrolysis-driven H+ translocation is stimulated by sulfate, a strong inhibitor of mitochondrial ATP synthesis

Sulfate is a partial inhibitor at low and a non-essential activator at high [ATP] of the ATPase activity of F(1). Therefore, a catalytically-competent ternary F(1) x ATP x sulfate complex can be formed. In addition, the ANS fluorescence enhancement driven by ATP hydrolysis in submitochondrial particles is also stimulated by sulfate, clearly showing that the ATP hydrolysis in its presence is coupled to H(+) translocation. However, sulfate is a strong linear inhibitor of the mitochondrial ATP synthesis. The inhibition was competitive (K (i) = 0.46 mM) with respect to Pi and mixed (K (i) = 0.60 and K'(i) = 5.6 mM) towards ADP. Since it is likely that sulfate exerts its effects by binding at the Pi binding subdomain of the catalytic site, we suggest that the catalytic site involved in the H(+) translocation driven by ATP hydrolysis has a more open conformation than the half-closed one (beta(HC)), which is an intermediate in ATP synthesis. Accordingly, ATP hydrolysis is not necessarily the exact reversal of ATP synthesis.     

Subunit interaction during catalysis. Alternating site cooperativity of mitochondrial adenosine triphosphatase.

ATP concentration modulates oxygen exchange catalyzed by purified, soluble mitochondrial ATPase during ATP hydrolysis so that water oxygen incorporation into each Pi formed increases markedly as ATP concentration is lowered. This behavior is readily explained by catalytic cooperativity between subunits of the ATPase. However, other reasonable explanations also need consideration. A new approach for assessing these various explanations is used, based on measurement of the [18O]Pi species formed by hydrolysis of ATP highly labeled with 18O in the gamma-phosphoryl group. The results and other supporting data give what appears to be the most compelling evidence yet attained for alternating site catalytic cooperativity in an enzymic catalysis.     

F1 ATPase from the thermophilic bacterium PS3 (TF1) shows ATP modulation of oxygen exchange

The ATPase from the ATP synthase of the thermophilic bacterium PS3 (TF1), unlike F1 ATPase from other sources, does not retain bound ATP, ADP, and Pi at a catalytic site under conditions for single-site catalysis [Yohda, M., & Yoshida, M. (1987) J. Biochem. 102, 875-883]. This raised a question as to whether catalysis by TF1 involved alternating participation of catalytic sites. The possibility remained, however, that there might be transient but catalytically significant retention of bound reactants at catalytic sites when the medium ATP concentration was relatively low. To test for this, the extent of water oxygen incorporation into Pi formed by ATP hydrolysis was measured at various ATP concentrations. During ATP hydrolysis at both 45 and 60 degrees C, the extent of water oxygen incorporation into the Pi formed increased markedly as the ATP concentration was lowered to the micromolar range, with greater modulation observed at 60 degrees C. Most of the product Pi formed arose by a single catalytic pathway, but measurable amounts of Pi were formed by a pathway with high oxygen exchange. This may result from the presence of some poorly active enzyme. The results are consistent with sequential participation of three catalytic sites on the TF1 as predicted by the binding change mechanism.     

Synthesis and hydrolysis of ATP and the phosphate-ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide.

In medium containing 40% dimethylsulfoxide, soluble F1 catalyzes the hydrolysis of ATP introduced at concentrations lower than that of the enzyme [Al-Shawi, M.K. & Senior, A.E. (1992), Biochemistry 31, 886-891]. At this concentration of dimethylsulfoxide, soluble F1 also catalyzes the spontaneous synthesis of a tightly bound ATP to a level of approximately 0.15 mol per mol F1 [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. The mechanisms that allow soluble F1 to carry out these apparently opposing reactions were studied. The rate of hydrolysis of ATP bound to F1 under uni-site conditions and that of synthesis of ATP were markedly similar, indicating that the two ATP molecules lie in equivalent high affinity catalytic sites. The number of enzyme molecules that have ATP at the high affinity catalytic site under conditions of synthesis or uni-site hydrolysis is less than the total number of enzyme molecules. Therefore, it was hypothesized that when the enzyme was treated with dimethylsulfoxide, a fraction of the F1 population carried out synthesis and another hydrolysis. Indeed, measurements of the two reactions under identical conditions showed that different fractions of the F1 population carried out simultaneously synthesis and hydrolysis of ATP. The reactions continued until an equilibrium level between F1.ADP + Pi <--> F1.ATP was established. At equilibrium, about 15% of the enzyme population was in the form F1.ATP. The DeltaG degrees of the reaction with 0.54 microM F1, 2 mM Pi and 10 mM Mg2+ at pH 6.8 was -2.7 kcal.mol-1 in favor of F1.ATP. The DeltaG degrees of the reaction did not exhibit important variations with Pi concentration; thus, the reaction was in thermodynamic equilibrium. In contrast, DeltaG degrees became significantly less negative as the concentration of dimethylsulfoxide was decreased. In water, the reaction was far to the left. The equilibrium constant of the reaction diminished linearly with an increase in water activity. The effect of solvent is fully reversible. In comparison to other enzymes, F1 seems unique in that solvent controls the equilibrium that exists within an enzyme population. This results from the effect of solvent on the partition of Pi between the catalytic site and the medium, and the large energetic barrier that prevents release of ATP from the catalytic site. In the presence of dimethylsulfoxide and Pi, ATP is continuously hydrolyzed and synthesized with formation and uptake of Pi from the medium. This process is essentially an exchange reaction analogous to the phosphate-ATP exchange reaction that is catalyzed by the ATP synthase in coupled energy transducing membranes.     

Properties of chloroplast F1-ATPase partially modified by 2-azido adenine nucleotides, including demonstration of three catalytic pathways

Previous investigations on the distribution of [18O]Pi isotopomers formed by hydrolysis of [gamma-18O]ATP by the chloroplast F1-ATPase (CF1) showed that a single reaction pathway is used by all participating sites and that the pathway is modulated by ATP concentration as expected for cooperative interactions between catalytic sites. Such oxygen exchange measurements have been applied to CF1 modified at a single catalytic or noncatalytic site by 2-azido adenine nucleotides. When less than one catalytic or one noncatalytic site per enzyme is modified, hydrolysis occurs in part by the pathway of the unmodified enzyme plus at least one additional pathway at 200 microM and two additional pathways at 4 microM [gamma-18O]ATP. Thus, three sites are potentially catalytically active. The two new pathways shown by the derivatized enzyme logically can arise from nonidentical interactions of the remaining two underivatized beta subunits with the derivatized beta subunit. Reversals of bound ATP cleavage before Pi is released are increased, and the amount of product formed by the new pathways is changed when the ATP concentration is lowered. These modulations must result from the behavior of two remaining active catalytic sites rather than of one catalytic and one regulatory site. When the CF1 is derivatized more extensively, the original catalytic pathway is lost, and two catalytic pathways that do not show modulation by ATP concentration are found. The remaining beta subunits now have weak but independent catalytic capacity. In addition, the enzyme is no longer activated by Ca2+, loses MgGTPase activity, and is much less sensitive to azide.     

Probes of catalytic site cooperativity during catalysis by the chloroplast adenosine triphosphate and the adenosine triphosphate synthase

During net nucleoside triphosphate synthesis by chloroplast ATP synthase the extent of water oxygen incorporation into each nucleoside triphosphate released increases with decrease in ADP, GDP or IDP concentration. Likewise, during net ATP hydrolysis by the Mg2+-activated chloroplast ATPase, the extent of water oxygen incorporation into each Pi released increases as the ATP, GTP, or ITP concentration is decreased. However, the concentration ranges in which substrate modulation occurs differs with each nucleotide. Modulation of oxygen exchange during synthesis and hydrolysis of adenine nucleotides, as measured by variation in the extent of water oxygen incorporation into products, occurs below 250 microM. In contrast, guanosine and inosine nucleotides alter the extent of exchange at higher and much wider concentration ranges. Activation of the chloroplast ATPase by either heat or trypsin results in similar catalytic behavior as monitored by ATP modulation of oxygen exchanges during hydrolysis in the presence of Mg2+. More exchange capacity is evident with octylglucoside-activated enzyme at all ATP concentrations. High levels of tentoxin were also found to alter the catalytic exchange parameters resulting in continued water oxygen exchange into Pi released during hydrolysis at high ATP concentrations. Little or no oxygen exchange accompanies ATP hydrolysis in the presence of Ca2+. The [18O]Pi species formed from highly gamma-18O-labeled ATP at lower ATP concentrations gives a distribution as expected if only one catalytic pathway is operative at a given ATP concentration. This and other results support the concept of catalytic cooperativity between alternating sites as explanation for the modulation of oxygen exchange by nucleotide concentration.     

Subunit interaction during catalysis. ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction

A new approach for assessing of catalytic cooperativity may occur between subunits has been applied to succinyl-CoA synthetase. This is based on the extent of oxygen exchange between medium [18O]Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis. Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium. With the Escherichia coli enzyme, which has an alpha 2 beta 2 structure, a pronounced increase in oxygen exchange per ATP cleaved occurs as ATP concentration is lowered. In contrast, when the CoA concentration is varied, the oxygen exchange per molecule of product formed remains constant. Also, with the pig heart enzyme, which is shown to retain its alpha beta structure during catalysis and thus has only one catalytic site, no modulation of oxygen exchange by ATP concentration is observed. These experimental findings show that the binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange. Measurement of the distribution of [18O]Pi species found as exchange occurs shows that only one catalytic sequence is involved in exchange at various ATP concentrations. These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at one catalytic site promotes catalytic site promotes catalytic events at an alternate catalytic site.     

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.     

ATP regulation of sarcoplasmic reticulum Ca2+-ATPase. Metal-free ATP and 8-bromo-ATP bind with high affinity to the catalytic site of phosphorylated ATPase and accelerate dephosphorylation

To localize and characterize the regulatory nucleotide site of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase, we have investigated the effects of ADP, ATP, and analogues of these nucleotides on the rate of dephosphorylation of both native ATPase and ATPase modified with fluorescein 5'-isothiocyanate (FITC), a reagent which hinders access of nucleotides to the ATPase catalytic site without affecting phosphorylation from Pi. Dephosphorylation of the phosphoenzyme formed from Pi was monitored by rapid filtration or stopped-flow fluorescence, mostly at 20 degrees C, pH 6.0, and in the absence of potassium. Fluorescence measurements were made possible through the use of 8-bromo-ATP, which selectively quenched certain tryptophan residues of the ATPase, thereby allowing the intrinsic fluorescence changes associated with dephosphorylation to be measured in the presence of bound nucleotide. ATP, 8-bromo-ATP, and trinitrophenyladenosine diand triphosphate, but not ADP, enhanced the rate of dephosphorylation of native ATPase 2-3-fold when added in the absence of divalent cations. Millimolar concentrations of Mg2+ eliminated the accelerating effects. Acceleration in the absence of Mg2+ was observed at relatively low concentrations of ATP and 8-bromo-ATP (0.01-0.1 mM) and binding of metal-free ATP and ADP, but not Mg.ATP, to the phosphoenzyme in this concentration range was demonstrated directly. Modification of the ATPase with FITC blocked nucleotide binding in the submillimolar concentration range and eliminated the nucleotide-induced acceleration of dephosphorylation. These results show that dephosphorylation, under these conditions, is regulated by ATP but not by Mg.ATP or ADP, and that the catalytic site is the locus of this "regulatory" ATP binding site.     

Interaction of adenosine-5'-O-(3-thiotriphosphate) with Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum

Interaction of adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) with Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. The nucleotide was slowly hydrolyzed by the ATPase at 30 degrees C at a rate of about 0.5% that of ATP hydrolysis. Whereas at 0 degrees C, ATP gamma S showed only a limited reactivity toward the ATPase in that a thiophosphorylated intermediate was formed and ADP was released, but hydrolysis of the intermediate to complete the catalytic cycle did not occur. A fairly stable analog of the E-P intermediate could thus be obtained. Presence of the thiophosphorylated intermediate was indicated by the [3H]ADP in equilibrium ATP gamma S exchange reaction and also by using [35S]ATP gamma S. When the ATPase was reacted with ATP gamma S at 0 degrees C in the presence of ferricyanide, EP-forming activity was rapidly lost. Free Ca2+ ions were required for this inactivation. Disulfide bond formation between a cysteinyl residue located near the substrate binding site and the enzyme-bound ATP gamma S or the thiophosphorylated intermediate was suggested by the fact that 2-mercaptoethanol reversed the inactivation. The reaction may prove to be a useful tool for affinity labeling of the active site of the ATPase.     

Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.