Alpha 3 beta 3 complex of thermophilic ATP synthase. Catalysis without the gamma-subunit

A complex of the alpha- and beta-subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the alpha beta gamma complex. The alpha 3 beta 3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa). Dilution of the alpha beta complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the gamma-subunit. A previous method using urea for isolating the subunits resulted in an alpha beta complex with lower activity than that prepared by over-expression of the genes. The alpha beta-ATP complex was formed from the alpha beta complex, ADP and Pi in the presence of dimethyl sulfoxide.     

The alpha 1 beta 1 heterodimer of ATP synthase

The alpha 3 beta 3 hexamer was reconstituted from the alpha and beta subunits of TF1 portion of ATP synthase of thermophilic bacterium (Kagawa et al. (1989) FEBS Lett. 249, 67). The alpha 1 beta 1 heterodimer of ATP synthase was isolated by high performance liquid chromatography (HPLC) of the alpha 3 beta 3 hexamer in the presence of AT(D)P-Mg. On polyacrylamide gel electrophoresis, both bands corresponding to the dimer and hexamer showed ATPase activity. The alpha 1 beta 1 dimer was dissociated into the equal amounts of the alpha and beta monomers by sodium dodecyl sulfate. The alpha and beta monomers were practically inactive. The alpha 2 and beta 2 homodimers were not detected by electrophoresis and HPLC.     

The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase

Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.     

Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase.

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies. The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site. Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced. Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding. When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+. When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent binding of 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) to the isolated alpha and beta subunits and the alpha 3 beta 3 core complex of TF1. Covalent binding of BzATP prevents association of alpha and beta subunits and induces dissociation of the alpha 3 beta 3 core complex.

Binding of the photoreactive ATP analog, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP), to the isolated alpha and beta subunits of TF1 and to the alpha 3 beta 3 "core" complex of the holoenzyme is described. About 1 mol of BzATP/mol of subunit was incorporated to isolated alpha and beta subunits. The incorporation of BzATP was prevented by ATP. Covalent binding of BzATP to the alpha subunit was in general somewhat lower than that observed with the beta subunit. No complex was formed upon mixing of either of the modified subunits with the complementary nontreated subunits. Covalent binding of 3 mol of BzATP/alpha 3 beta 3 complex completely inhibited ATPase activity and resulted in the dissociation of the complex. The labeled nucleotide analog was specifically incorporated into the beta subunit of the complex. The holoenzyme TF1, in contrast to the core complex, did not dissociate to the individual subunits upon covalent binding of BzATP. These results are discussed in relation to the location of the catalytic nucleotide binding site(s) and the conformation stability of the alpha 3 beta 3 core complex of TF1.     

Origin of apparent negative cooperativity of F(1)-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F(1)-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F(1)-ATPase, alpha((W463F)3)beta((Y341W)3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent K(m)'s of ATPase kinetics (4.0 and 233 microM) did not agree with apparent K(m)'s deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 microM). On the other hand, in case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K(m) of ATPase activity (10 microM) roughly agreed with the highest K(m) of fluorescence measurements (27 microM). The results indicate that in case of alpha((W463F)3)beta((Y341W)3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K(m) of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, the K(m) of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W)3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F(1)-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K(m)'s of 100-300 microM and 1-30 microM range for wild-type F(1)-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

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.     

Cryoenzymic studies on actomyosin ATPase: kinetic evidence for communication between the actin and ATP sites on myosin.

The post-ATP binding steps of myosin subfragment 1 (S1) and actomyosin subfragment 1 (actoS1) ATPases were studied at -15 degrees C with 40% ethylene glycol as antifreeze. The cleavage and release of Pi steps were studied by the rapid-flow quench method and the interaction of actin with S1 plus ATP by light scattering in a stopped-flow apparatus. At -15 degrees C, the interaction of actin with S1 remains tight, and the Km for the activation of S1 ATPase is very small (0.3 microM). The chemical data were interpreted by E + ATP----E*.ATP----E**.ADP.Pi----E*.ADP----products, where E is S1 or actoS1. In Pi burst experiments with S1, there was a large Pi burst of free Pi, but E**.ADP.Pi could not be detected. Here the predominant complex in the seconds time range is E*.ATP and in the steady-state E*.ADP. With actoS1, there was a small Pi burst of E**.ADP.Pi, evidence that the cleavage steps for S1 and actoS1 are different. From the stopped-flow experiments, the dissociation of actoS1 by ATP was complete, even at actin concentrations 60X its Km. Further, no interaction of actin with the key intermediate M*.ATP could be detected. Therefore, at -15 degrees C, actoS1 ATPase occurs by a dissociative pathway; in particular, the cleavage step appears to occur in the absence of actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The noncatalytic site-deficient alpha3beta3gamma subcomplex and FoF1-ATP synthase can continuously catalyse ATP hydrolysis when Pi is present.

We investigated ATP hydrolysis by a mutant (DeltaNC) alpha3beta3gamma subcomplex of F0F1-ATP synthase from the thermophilic Bacillus PS3 that is defective in the noncatalytic nucleotide binding sites. This mutant subcomplex was activated by inorganic phosphate ions (Pi) and did not show continuous ATP hydrolysis activity in the absence of Pi. Pi also activated the wild-type alpha3beta3gamma subcomplex in a similar manner. Sulphate activated wild-type alpha3beta3gamma but not DeltaNC alpha3beta3gamma, indicating that Pi activation did not involve noncatalytic sites but that sulphate activation did. Pi also activated ATP hydrolysis and coupled proton translocation by the wild-type and DeltaNC F0F1-ATP synthases reconstituted into vesicle membranes.     

Enzyme phosphorylation with inorganic phosphate causes Ca2+ dissociation from sarcoplasmic reticulum adenosinetriphosphatase

Sarcoplasmic reticulum ATPase is phosphorylated by ATP in the presence of calcium, with a consequent reduction of the affinity of the binding sites for calcium and dissociation of the divalent cation from the enzyme. ATPase phosphorylation with Pi, on the other hand, requires prior removal of calcium from the enzyme, indicating that the energy requirement for phosphorylation of the enzyme-calcium complex can be met by ATP but not by Pi. We find that when the energy yield of the Pi reaction with the enzyme is increased by the addition of dimethyl sulfoxide to the medium, ATPase phosphorylation with Pi occurs even in the presence of calcium, and the binding sites undergo a reduction in affinity with consequent dissociation of Ca2+ from the enzyme, in analogy to the effect of ATP. It is thereby demonstrated experimentally that an essential step in the coupling of catalytic and transport activities is an interdependence and mutual ligand exclusion of the phosphorylation and calcium sites, in which ATP does not play a direct role. An important difference between the effects of ATP and Pi is that the former produces dissociation of Ca2+ inside the vesicles as the result of advancement of the catalytic cycle in the forward direction, while Pi produces dissociation of calcium into the outer medium as a consequence of equilibration of enzyme states producing a shift in the reverse direction of the enzyme cycle. These observations demonstrate how equilibration of intermediate enzyme states determines extent and direction of overall reaction flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the catch-loop tyrosine beta Y317 with the metal at catalytic site 3 of Chlamydomonas chloroplast F1-ATPase.

Site-directed mutants Y317C, Y317E, Y317F, Y317G, and Y317K were made to the catch-loop tyrosine on the beta subunit of the chloroplast F(1)-ATPase in Chlamydomonas. EPR spectra of VO(2+)-ATP bound to site 3 of CF(1) from wild type and mutants were obtained. Every mutant changed the (51)V hyperfine parameters of the VO(2+) bound at this site in the catalytically active conformation of the enzyme but had no effect on these parameters in the form that predominates when the enzyme activity is latent. These results indicate that this residue is a ligand to the metal of the Mg(2+)-nucleotide complex that binds to the empty catalytic site. The mutations also decreased the k(cat) of the ATPase activity to a much greater extent than k(cat)/K(M). Thus, these mutations limit the rate of product (Mg(2+)-ADP and phosphate) release in the ATPase direction or, conversely, the initial binding of substrates in the ATP synthesis direction. On the basis of these observations, coordination of betaY317 by Mg(2+)-ADP that binds to the empty catalytic site provides a means by which substrate binding could trigger gamma subunit rotation and consequent conformation changes of beta subunits during ATP synthesis.     

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.     

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.     

Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases

This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigation of the substrate structure and metal cofactor requirements of the rat liver mitochondrial ATP synthase/ATPase complex

The F1 moiety of the rat liver mitochondrial ATP synthase/ATPase complex contains as isolated 2 mol Mg2+/mol F1, 1 mol of which is nonexchangeable and the other which is exchangeable (N. Williams, J. Hullihen, and P.L. Pedersen, (1987) Biochemistry 26, 162-169). In addition, the enzyme binds 1 mol ADP/mol F1 and 3 mol AMP.PNP, the latter of which can bind in complex formation with divalent cation and displace the Mg2+ at the exchangeable site. Thus, in terms of ligand binding sites the fully loaded rat liver F1 complex contains 3 mol MgAMP.PNP, 1 mol ADP, and 1 mol Mg2+. In this study we have used several metal ATP complexes or analogs thereof to gain further insight into the ligand binding domains of rat liver F1 and the mechanism by which it catalyzes ATP hydrolysis in soluble and membrane bound form. Studies with LaATP confirmed that MgATP is the most likely substrate for rat liver F1, and provided evidence that the enzyme may contain additional Mg2+ binding sites, undetected in previous studies of F1-ATPases, that are required for catalytic activity. Thus, F1 containing the thermodynamically stable LaATP complex in place of MgATP requires added Mg2+ to induce ATP hydrolysis. As Mg2+ cannot readily displace La2+ under these conditions there appears to be a catalytically important class of Mg2+ binding sites on rat liver F1, distinct from the nonexchangeable Mg2+ site and the sites involved in binding MgATP. Additional studies carried out with exchange inert metal-nucleotide complexes involving rhodium and the Mg2+ and Cd2+ complexes of ATP beta S and ATP alpha S imply that the rate-limiting step in the ATPase reaction pathway occurs subsequent to the P gamma-O-P beta bond cleavage steps, perhaps at the level of Mg(ADP)(Pi) hydrolysis or MgADP release. Evidence is presented that Mg2+ remains coordinated to the leaving group of the reaction, i.e., the beta phosphoryl group. Finally, in contrast to soluble F1, F1 bound to F0 in the inner mitochondrial membrane failed to discriminate between the Mg2+ complexes of the ATP beta S isomers. This indicates that a fundamental difference may exist between the catalytic or kinetic mechanism of F1 and the more physiologically intact F0F1 complex.     

Mitochondrial ATP synthase: dramatic Mg2+-induced alterations in the structure and function of the F1-ATPase moiety

The ATPase activity of the F1 moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 degrees C in the presence of MgCl2. The concentration of MgCl2 (130 microM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1 complex of alpha subunits and part of the gamma-subunit population. The latter form a precipitate while the beta, delta, and epsilon subunits, and the remaining part of the gamma-subunit population, remain soluble. Dissociation is not a sudden "all or none" event but parallels loss of ATPase activity until alpha subunits have almost completely dissociated together with about 50% of the gamma-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPi at high concentration (greater than or equal to 200 mM) is effective in partially protecting F1 against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0 moiety in F1-depleted particles. When bound to F0, F1 is protected completely against divalent cation induced inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal ion dependence of phosphorothioate ATP analogues in the Bacillus stearothermophilus tyrosyl-tRNA synthetase reaction

Pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of alpha- and beta-phosphorothioate adenosine triphosphates [ATP alpha S and ATP beta S; Eckstein, F., & Goody, R. (1976) Biochemistry 15, 1685-1691; Yee, D., Armstrong, V. W., & Eckstein, F. (1979) Biochemistry 18, 4116-4123] were performed in the presence of Mg2+, Co2+, and Cd2+ as the divalent metal ion cofactor. A modest preference of 5.5-fold in kappa 3/KA' (where kappa 3 is the rate constant for tyrosyl adenylate formation and KA' is the dissociation constant for ATP, or phosphorothioate ATP, from the E.Tyr.metal.ATP complex) for the Sp ATP alpha S diastereomer and the absence of an inversion of preference when the metal ion is changed suggest that there is a stereospecific enzyme-alpha-phosphate interaction and that there is no direct metal ion interaction with the alpha-phosphate. The extent of reaction of the ATP alpha S diastereomers (30-50%) implies that these analogues are more susceptible to the hydrolytic site reaction previously reported for this enzyme [Wells, T. N. C., & Fersht, A. R. (1986) Biochemistry 25, 1881-1886]. The strong preference in kappa 3/KA' for the RP ATP beta S diastereomer (16-fold for Mg2+ and 50-fold for Co2+) is indicative of a stereospecific interaction with the pro SP beta oxygen of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-linking of the gamma subunit of the Escherichia coli ATPase (ECF1) via cysteines introduced by site-directed mutagenesis.

The gamma subunit of the Escherichia coli F1 ATPase (ECF1) has been altered by site-directed mutagenesis to create five different mutants, gamma-S8C, gamma-S81C, gamma-T106C, gamma-S179C, and gamma-V286C, respectively. ECF1 isolated from four of these mutants had ATPase activities similar to that of a wild-type isogenic strain used as a control, the exception was enzyme isolated from mutant gamma-S81C, which had an ATPase activity of around 70-80% of the wild type. ECF1 isolated from each of the various mutants was reacted with N-(4-(7-(diethylamino)-4-methylcoumarin-3-yl))maleimide (CM). The fluorescent reagent was incorporated into Cys residues placed at positions 8, 106, 179, and 286, but not at 81, indicating which of these Cys residues are on the surface of the gamma subunit in the enzyme complex. Modification of the Cys at position 106 with CM activated the enzyme, and modification of the Cys at position 8 inhibited ATPase activity a small amount; however, modification of Cys at 179 or 286 had no effect on enzyme activity. The four mutants with a reactive Cys were reacted with tetrafluorophenylazide maleimides (TFPAMs), novel photoactivatable cross-linkers. In the mutant gamma-S8C, cross-links were formed between the introduced Cys on the gamma subunit and sites on the beta subunit. This cross-linking between gamma and beta depended on nucleotide conditions under which the photolysis was carried out, with differently migrating cross-linked products being obtained in ATP + EDTA compared with ATP + Mg2+ or ATP + Mg2+ Pi. Cross-linking between beta and gamma inhibited ATPase activity in proportion to the yield of cross-linked product. In the mutant gamma-V286C, cross-links were formed between the introduced Cys on gamma and the alpha subunit which were the same in all nucleotide conditions and which led to inhibition of ATPase activity.     

Reversal of the sarcoplasmic reticulum ATPase cycle by substituting various cations for magnesium. Phosphorylation and ATP synthesis when Ca2+ replaces Mg2+

Reversal of the cycle of sarcoplasmic reticulum ATPase starts from ATPase phosphorylation by Pi, in the presence of Mg2+, and leads to ATP synthesis. We show here that ATP can also be synthesized when Ca2+ replaces Mg2+. In the absence of a calcium gradient and in the presence of dimethyl sulfoxide, ATPase phosphorylation from Pi and Ca2+ led to the formation of an unstable phosphoenzyme. This instability was due to a competition between the phosphorylation reaction induced by Pi and Ca2+ and the transition induced by Ca2+ binding to the transport sites, which led to a conformation that could not be phosphorylated from Pi. Dimethyl sulfoxide and low temperature stabilized the calcium phosphoenzyme, which under appropriate conditions, subsequently reacted with ADP to synthesize ATP. Substitution of Co2+, Mn2+, Cd2+, or Ni2+ for Mg2+ induced ATPase phosphorylation from Pi, giving phosphoenzymes of various stabilities. However, substitution of Ba2+, Sr2+, or Cr3+ produced no detectable phosphoenzymes, under the same experimental conditions. Our results show that ATPase phosphorylation from Pi, like its phosphorylation from ATP, does not have a strict specificity for magnesium.     

Iron(III)-mediated photolysis of outer arm dynein ATPase from sea urchin sperm flagella

Irradiation of outer arm dynein ATPase from sea urchin sperm tail flagella at 365-410 nm in the presence of Fe(III)-gluconate complex and ATP produces photolytic cleavage at two distinct sites on the beta heavy chain, located approximately 250 and approximately 230 kDa from its amino terminus. The former cut is close to or identical with the V1 site of the vanadate-mediated photocleavage (Gibbons, I.R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J.Y., and Gibbons, B.H. (1987) J. Biol. Chem. 262, 2780-2786. The rate of photolysis shows a hyperbolic dependence on Fe(III)-gluconate concentration with half-maximal rate occurring at 23 microM at pH 6.3. In the presence of 0.1-0.5 mM Fe(III)-gluconate-ATP, approximately 58% of the beta chain becomes cleaved with a half-time of about 34 s; the remainder of the beta chain and almost all of the alpha chain are resistant to cleavage. This photolytic cleavage of the beta chain is accompanied by an approximately parallel loss of the dynein latent ATPase activity, whereas the Triton-activated ATPase is lost to a somewhat greater extent. Mg2+ concentrations above approximately 3 mM inhibit photolysis. Substitution of ADP for ATP changes the pattern of cleavage so that both the alpha and beta heavy chain undergo scission but at the 250-kDa site only. AMP, adenyl-5'-yl imidodiphosphate and Fe(II) do not support cleavage at either site. Trivalent rhodium-ATP complexes, as models of MgATP, can also catalyze photolysis of the beta chain at the 250-kDa site. These results suggest that photolysis results from the activation of an Fe(III)-ATP complex bound to the hydrolytic ATP binding site of the beta chain and that both Fe(III) cleavage sites are located close to the nucleotide binding site in the tertiary folding of the beta heavy chain. The cleavage reaction possibly involves initial photoreduction of Fe(III) bound at the Mg2+ binding site in the dynein.Fe.ATP complex, followed by covalent modification of an amino acid side chain that leads to eventual peptide scission.