Rotary catalysis of the stator ring of F(1)-ATPase

F(1)-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α(3)β(3) ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a "dictator" completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F(1)-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F₁-ATPase (F₁) by attaching probes with different viscous drag coefficients at the rotary shaft of F₁. Individual rotation pauses of F₁ between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F₁ rate enhancement.     

Robustness of the Rotary Catalysis Mechanism of F1-ATPase

F₁-ATPase (F₁) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F₁ as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F₁ is far more robust than previously thought.     

Determination of the partial reactions of rotational catalysis in F1-ATPase

Steady-state ATP hydrolysis in the F1-ATPase of the F(O)F1 ATP synthase complex involves rotation of the central gamma subunit relative to the catalytic sites in the alpha3beta3 pseudo-hexamer. To understand the relationship between the catalytic mechanism and gamma subunit rotation, the pre-steady-state kinetics of Mg x ATP hydrolysis in the soluble F1-ATPase upon rapid filling of all three catalytic sites was determined. The experimentally accessible partial reactions leading up to the rate-limiting step and continuing through to the steady-state mode were obtained for the first time. The burst kinetics and steady-state hydrolysis for a range of Mg x ATP concentrations provide adequate constraints for a unique minimal kinetic model that can fit all the data and satisfy extensive sensitivity tests. Significantly, the fits show that the ratio of the rates of ATP hydrolysis and synthesis is close to unity even in the steady-state mode of hydrolysis. Furthermore, the rate of Pi binding in the absence of the membranous F(O) sector is insignificant; thus, productive Pi binding does not occur without the influence of a proton motive force. In addition to the minimal steps of ATP binding, reversible ATP hydrolysis/synthesis, and the release of product Pi and ADP, one additional rate-limiting step is required to fit the burst kinetics. On the basis of the testing of all possible minimal kinetic models, this step must follow hydrolysis and precede Pi release in order to explain burst kinetics. Consistent with the single molecule analysis of Yasuda et al. (Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., and Itoh, H. (2001) Nature 410, 898-904), we propose that the rate-limiting step involves a partial rotation of the gamma subunit; hence, we name this step k(gamma). Moreover, the only model that is consistent with our data and many other observations in the literature suggests that reversible hydrolysis/synthesis can only occur in the active site of the beta(TP) conformer (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628).     

Structural model of F1-ATPase and the implications for rotary catalysis

The crystal structure of bovine mitochondrial F1-ATPase is described. Several features of the structure are consistent with the binding change mechanism of catalysis, in which binding of substrates induces conformational changes that result in a high degree of cooperativity between the three catalytic sites. Furthermore, the structure also suggests that catalysis is accompanied by a physical rotation of the centrally placed gamma-subunit relative to the approximately spherical alpha3beta3 subassembly.     

Principal role of the arginine finger in rotary catalysis of F1-ATPase

F(1)-ATPase (F(1)) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α(3)β(3) stator ring. The 3 catalytic sites of F(1) reside on the interface of the α and β subunits of the α(3)β(3) ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F(1) remains controversial. We studied the role of the arginine finger by analyzing the rotation of a mutant F(1) with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F(1) showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F(1) carrying a single copy of the α mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATP-binding rate.     

F0F1-ATPase gamma subunit mutations perturb the coupling between catalysis and transport.

We introduced mutations to test the function of the conserved amino-terminal region of the gamma subunit from the Escherichia coli ATP synthase (F0F1-ATPase). Plasmid-borne mutant genes were expressed in an uncG strain which is deficient for the gamma subunit (gamma Gln-14-->end). Most of the changes, which were between gamma Ile-19 and gamma Lys-33, gamma Asp-83 and gamma Cys-87, or at gamma Asp-165, had little effect on growth by oxidative phosphorylation, membrane ATPase activity, or H+ pumping. Notable exceptions were gamma Met-23-->Arg or Lys mutations. Strains carrying these mutations grew only very slowly by oxidative phosphorylation. Membranes prepared from the strains had substantial levels of ATPase activity, 100% compared with wild type for gamma Arg-23 and 65% for gamma Lys-23, but formed only 32 and 17%, respectively, of the electrochemical gradient of protons. In contrast, other mutant enzymes with similar ATPase activities (including gamma Met-23-->Asp or Glu) formed H+ gradients like the wild type. Membranes from the gamma Arg-23 and gamma Lys-23 mutants were not passively leaky to protons and had functional F0 sectors. These results suggested that substitution by positively charged side chains at position 23 perturbed the energy coupling. The catalytic sites of the mutant enzymes were still regulated by the electrochemical H+ gradient but were inefficiently coupled to H+ translocation in both ATP-dependent H+ pumping and delta mu H+ driven ATP synthesis.     

A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis

The F(0)F(1)-ATP synthase couples the functions of H(+) transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located gamma, epsilon, and c subunits. To understand the gamma subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F(1)-ATPase enzyme that uniquely fits the pre-steady state and steady state data ( Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797 ). Here we directly test the model using two single cysteine mutants, betaD380C and betaE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved gammaCys-87. In the pre-steady state, the gamma-beta cross-linked enzyme at high Mg.ATP conditions retained the burst of hydrolysis but was not able to release P(i). These data show that the rate-limiting rotation step, k(gamma), occurs after hydrolysis and before P(i) release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the betaGlu-381 residue for proper formation of the rate-limiting transition state involving gamma subunit rotation.     

Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase.

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.     

Rotary F1-ATPase. Is the C-terminus of subunit gamma fixed or mobile?

F-ATP synthase synthesizes ATP at the expense of ion motive force by a rotary coupling mechanism. A central shaft, subunit gamma, functionally connects the ion-driven rotary motor, F(O), with the rotary chemical reactor, F(1). Using polarized spectrophotometry we have demonstrated previously the functional rotation of the C-terminal alpha-helical portion of gamma in the supposed 'hydrophobic bearing' formed by the (alpha beta)(3) hexagon. In apparent contradiction with these spectroscopic results, an engineered disulfide bridge between the alpha-helix of gamma and subunit alpha did not impair enzyme activity. Molecular dynamics simulations revealed the possibility of a 'functional unwinding' of the alpha-helix to form a swivel joint. Furthermore, they suggested a firm clamping of that part of gamma even without the engineered cross-link, i.e. in the wild-type enzyme. Here, we rechecked the rotational mobility of the C-terminal portion of gamma relative to (alpha beta)(3). Non-fluorescent, engineered F(1) (alpha P280C/gamma A285C) was oxidized to form a (nonfluorescent) alpha gamma heterodimer. In a second mutant, containing just the point mutation within alpha, all subunits were labelled with a fluorescent dye. Following disassembly and reassembly of the combined preparations and cystine reduction, the enzyme was exposed to ATP or 5'-adenylyl-imidodiphosphate (AMP-PNP). After reoxidation, we found fluorescent alpha gamma dimers in all cases in accordance with rotary motion of the entire gamma subunit under these conditions. Molecular dynamics simulations covering a time range of nanoseconds therefore do not necessarily account for motional freedom in microseconds. The rotation of gamma within hours is compatible with the spectroscopically detected blockade of rotation in the AMP-PNP-inhibited enzyme in the time-range of seconds.     

The structure of bovine mitochondrial F1-ATPase: an example of rotary catalysis

There is now compelling evidence in support of a rotary catalytic mechanism in F1-ATPase, and, by extension, in the intact ATP synthase. Although models have been proposed to explain how protein translocation in F0 results in rotation of the gamma-subunit relative to the alpha 3/beta 3 assembly in F1 [22], these are still speculative. It seems likely that a satisfactory explanation of this mechanism will ultimately depend on structural information on the intact ATP synthase.     

Uni-site catalysis by Escherichia coli F1-ATPase with different numbers of bound nucleotides

We prepared two types of E. coli F1 by slightly different gel filtration procedures of the purified F1: F1(II) contained about 2 mol, and F1(V) about 5 mol of bound adenine nucleotides per mol of the enzyme. Thus F1(II) had more than 2, possibly 3, vacant catalytic sites, while F1(V) had less than one vacant catalytic site. The rate of ATP hydrolysis in uni-site catalysis (in the presence of inorganic phosphate) was about 3-fold higher with F1(II) than with F1(V), suggesting that ADP and inorganic phosphate bound at the catalytic sites of F1(V) changed the kinetics of uni-site catalysis significantly.     

Chaperones of F1-ATPase

Mitochondrial F₁-ATPase contains a hexamer of alternating α and β subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to β and α. In the absence of Atp11p and Atp12p, the hexamer is not formed, and α and β precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F₁ assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486–1493) hypothesized that the chaperones themselves look very much like the α and β subunits, and proposed an exchange of Atp11p for α and of Atp12p for β; the driving force for the exchange was expected to be a higher affinity of α and β for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to β and Atp12p is bound to α, the two F₁ subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to α and β prevents further interactions between these F₁ subunits. However, Atp11p and Atp12p do not resemble α or β, and it is instead the F₁ γ subunit that initiates the release of the chaperones from α and β and their further assembly into the mature complex.Mitochondrial F₁-ATPase consists of three α and three β subunits occupying alternate positions in a hexamer that surrounds a rod-like element containing one each of γ, δ, and ϵ subunits (1–3). Three nucleotide-binding catalytic sites (CS)⁴ and three noncatalytic sites (NCS) alternate at the six α/β interfaces. Early work with respiratory-deficient strains of Saccharomyces cerevisiae (4) revealed that two additional mitochondrial proteins, Atp11p and Atp12p, which are not integral subunits of the enzyme, are nonetheless necessary for the assembly of F₁-ATPase. Besides their failure to assemble F₁, a particularly interesting feature of atp11 and atp12 mutants is that they accumulate α and β subunits as high molecular weight aggregates (4) that can be recognized as densely stained inclusion bodies in the mitochondrial matrix (5). Subsequent studies in yeast have shown that Atp12p binds to F₁ α (6) and that Atp11p binds to β (7); these interactions include binding determinants in the nucleotide binding domains (NBD) of the two F₁ subunits. On this basis, it is now recognized that Atp11p and Atp12p are members of two new families of molecular chaperones, pfam06644 and pfam07542 (8), which are required for the assembly of mitochondrial ATP synthase in all eukaryotes. In fact, the first nuclear genetic lesion associated to a defect of mitochondrial ATP synthase in humans was identified in the locus ATPAF2 for Atp12p and was responsible for the death of a 14-month-old infant (9). Atp12p is also present in the α subdivision of Proteobacteria, consistent with the proposed origin of mitochondria from this ancestral line (10).The nature of the interactions between the F₁ subunits and Atp11p and Atp12p has remained elusive because of the lack of structural information for these chaperones. As α and β aggregate in the absence of Atp11p and Atp12p, it is usually assumed that the F₁ subunits are themselves poorly soluble, and that the two chaperones maintain them in a dispersed state until they are incorporated in the mature enzyme. Based on the analysis of the distribution of hydrophilic and hydrophobic areas on the surface of the α and β subunits of F₁, and on the interaction energies between these subunits at the interfaces that provide the CS and NCS sites, Wang et al. (6) have proposed a model of F₁ assembly in which Atp11p binds at the region of the β subunit that contributes to the CS site, and Atp12p binds at the region of the α subunit that contributes to the NCS site. One consequence of this particular binding of Atp11p and Atp12p to the F₁ subunits is that as long as Atp11p is bound to β and Atp12p is bound to α, the two F₁ subunits cannot interact at either the CS or the NCS interface. Since no other modulators of chaperone release are known, the Wang model requires an exchange of Atp11p for α and of Atp12p for β. Implied in this model is that the chaperones must themselves look very much like the α and β subunits, and that the driving force for the exchange must simply be a higher affinity of α and β for each other than for the respective chaperone partners. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to α and β prevents further interactions between these F₁ subunits. However, Atp11p and Atp12p do not resemble α or β, and it is instead the F₁ γ subunit that initiates the release of the chaperones from α and β and their further assembly into mature complex.     

Structural comparison of F1-ATPase: interplay among enzyme structures, catalysis, and rotations

F(1)-ATPase, a rotary motor powered by adenosine triphosphate hydrolysis, has been extensively studied by various methods. Here, we performed a systematic comparison of 29 X-ray crystal structures of F(1)-complexes, finding fine interplay among enzyme structures, catalysis, and rotations. First, analyzing the 87 structures of enzymatic αβ-subunits, we confirmed that the two modes, the hinge motion of β-subunit and the loose/tight motion of the αβ-interface, dominate the variations. The structural ensemble was nearly contiguous bridging three clusters, αβ(TP), αβ(DP), and αβ(E). Second, the catalytic site analysis suggested the correlation between the phosphate binding and the tightening of the αβ-interface. Third, addressing correlations of enzymatic structures with the orientations of the central stalk γ, we found that the γ rotation highly correlates with loosening of αβ(E)-interface and β(DP) hinge motions. Finally, calculating the helix 6 angle of β, we identified the recently observed partially closed conformation being consistent with β(HC).     

Single-site catalysis of F1-ATPase from thermophilic bacterium PS3 and its dominance in steady-state catalysis at low ATP concentration

Single-site catalysis by F1-ATPase from a thermophilic bacterium PS3 (TF1) was examined by incubating the enzyme with a submolar amount of radioactive ATP. The profile of single-site catalysis by TF1 at 23 degrees C was different from that of beef heart mitochondrial F1-ATPase (MF1). ATP hydrolysis on the enzyme and release of the products was rapid, and subsequent addition of non-radioactive ATP (cold chase) did not promote the hydrolysis of radioactive ATP, indicating that the rate-limiting step was not the step of product release but the step of ATP binding to the enzyme. Thus, the characteristic features of so-called uni-site catalysis were not observed. At 60 degrees C, whether in the presence or absence of phosphate ion, a small amount of bound [alpha, gamma-32P]ATP and cold chase promotion were observed. However, since bound 32P1 was not detected by centrifugal gel filtration, it is not yet certain whether TF1 has typical uni-site characteristics. Based on the hydrolytic turnover rate for single-site catalysis and analysis of the kinetics of steady-state catalysis, it is proposed that single-site catalysis is dominant even in steady-state catalysis at ATP concentrations of less than about 20 microM.     

Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Torque generation in F1-ATPase devoid of the entire amino-terminal helix of the rotor that fills half of the stator orifice

F₁-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α₃β₃ subunits. The amino and carboxyl termini of the γ rotor form a coiled coil of α-helices that penetrates the stator cylinder to serve as an axle. Crystal structures indicate that the axle is supported by the stator at two positions, at the orifice and by the hydrophobic sleeve surrounding the axle tip. The sleeve contacts are almost exclusively to the longer carboxyl-terminal helix, whereas nearly half the orifice contacts are to the amino-terminal helix. Here, we truncated the amino-terminal helix stepwise up to 50 residues, removing one half of the axle all the way up and far beyond the orifice. The half-sliced axle still rotated with an unloaded speed a quarter of the wild-type speed, with torque nearly half the wild-type torque. The truncations were made in a construct where the rotor tip was connected to a β-subunit via a short peptide linker. Linking alone did not change the rotational characteristics significantly. These and previous results show that nearly half the normal torque is generated if rotor-stator interactions either at the orifice or at the sleeve are preserved, suggesting that the make of the motor is quite robust.     

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH.

Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-32P]ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound [32P]Pi equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature-induced states of isolated F1-ATPase affect catalysis, enzyme conformation and high-affinity nucleotide binding sites

Isolated, nucleotide-depleted bovine-heart F1-ATPase exhibits a break in Arrhenius plot with a 2.7-fold increase in activation energy of ATP hydrolysis below 18-19 degrees C. Analysis of intrinsic tyrosine fluorescence and of the circular dichroism of F1-ATPase showed an abrupt and reversible conformational change occurring at the break temperature, characteristic of a structural tightening at low temperature. Analysis of catalytic nucleotide binding sites using fluorescent ADP analog, 3'-O-(1-naphthoyl)adenosine diphosphate did not show any significant change in affinity of nucleotide binding around the transition temperature but the bound fluorophore exerted a more restricted motion and slower rotation at temperature below the break, indicating a change in the mobility of groups in the close neighbourhood. It is concluded that, as a result of temperature, two kinetically distinct states of F1-ATPase are induced, due to a change in enzyme conformation, which influences directly the properties of catalytic nucleotide binding sites.     

Coupling of rotation and catalysis in F(1)-ATPase revealed by single-molecule imaging and manipulation

F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.