Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase

Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F1-ATPase.     

The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F(1)-ATPase.

F1-ATPase is inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or P(i). Here, using a mutant alpha(3)beta(3)gamma complex of thermophilic F(1)-ATPase (alpha W463F/beta Y341W) and monitoring nucleotide binding by fluorescence quenching of an introduced tryptophan, we found that P(i) interfered with the binding of MgATP to F(1)-ATPase, but binding of MgADP was interfered with to a lesser extent. Hydrolysis of MgATP by F(1)-ATPase during the experiments did not obscure the interpretation because another mutant, which was able to bind nucleotide but not hydrolyse ATP (alpha W463F/beta E190Q/beta Y341W), also gave the same results. The half-maximal concentrations of P(i) that suppressed the MgADP-inhibited form and interfered with MgATP binding were both approximately 20 mm. It is likely that the presence of P(i) at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme-MgADP-P(i) complex, an active intermediate in the catalytic cycle.     

Importance of F1-ATPase residue alpha-Arg-376 for catalytic transition state stabilization

The functional role of essential residue alpha-Arg-376 in the catalytic site of F1-ATPase was studied. The mutants alpha R376C, alpha R376Q, and alpha R376K were constructed, and combined with the mutation beta Y331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis. Despite the apparent proximity of alpha-Arg-376 to bound nucleoside di- and triphosphate in published X-ray structures, the mutations had little effect on MgADP or MgATP binding affinities, particularly at the highest affinity catalytic site, site 1. Both Cys and Gln mutants abolished transition state formation, demonstrating that alpha-Arg-376 is normally involved at this step of catalysis. A model of the F1-ATPase catalytic transition state structure is presented and discussed. The Lys mutant, although severely impaired, supported transition state formation, suggesting that an additional essential role for the alpha-Arg-376 guanidinium group exists, likely in alpha/beta conformational signal transmission required for steady-state catalysis. Parallels between alpha-Arg-376 and GAP/G-protein "arginine finger" residues are evident.     

Conserved walker A Ser residues in the catalytic sites of P-glycoprotein are critical for catalysis and involved primarily at the transition state step

P-glycoprotein mutants S430A/T and S1073A/T, affecting conserved Walker A Ser residues, were characterized to elucidate molecular roles of the Ser and functioning of the two P-glycoprotein catalytic sites. Results showed the Ser-OH is critical for MgATPase activity and formation of the normal transition state, although not for initial MgATP binding. Mutation to Ala in either catalytic site abolished MgATPase and transition state formation in both sites, whereas Thr mutants had similar MgATPase to wild-type. Trapping of 1 mol of MgADP/mol of P-glycoprotein by vanadate, shown here with pure protein, yielded full inhibition of ATPase. Thus, congruent with previous work, both sites must be intact and must interact for catalysis. Equivalent mutations (Ala or Thr) in the two catalytic sites had identical effects on a wide range of activities, emphasizing that the two catalytic sites function symmetrically. The role of the Ser-OH is to coordinate Mg(2+) in MgATP, but only at the stage of the transition state are its effects tangible. Initial substrate binding is apparently to an "open" catalytic site conformation, where the Ser-OH is dispensable. This changes to a "closed" conformation required to attain the transition state, in which the Ser-OH is a critical ligand. Formation of the latter conformation requires both sites; both sites may provide direct ligands to the transition state.     

Effect of the epsilon-subunit on nucleotide binding to Escherichia coli F1-ATPase catalytic sites.

The influence of the epsilon-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (beta-Trp-331). The interaction between epsilon and F1 was not affected by the mutation. Kd for binding of epsilon to betaY331W mutant F1 was approximately 1 nM, and epsilon inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the epsilon-depleted and epsilon-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. Kd1(MgATP) and Kd1(MgADP) were an order of magnitude higher in the absence of epsilon than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the epsilon-depleted and epsilon-replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of epsilon, Km equals Kd3. Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (Vmax) MgATP hydrolysis rates.     

The binding mechanism of the yeast F1-ATPase inhibitory peptide: role of catalytic intermediates and enzyme turnover

The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.     

The role of beta-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase.

Beta-Arg-182 in Escherichia coli F1-ATPase (beta-Arg-189 in bovine mitochondrial F1) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al. (1994) Nature 370, 621-628). Here we investigated the role of this residue by characterizing two mutants, betaR182Q and betaR182K. Oxidative phosphorylation and steady-state ATPase activity of purified F1 were severely impaired by both mutations. Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of beta-Trp-331 that occurred upon nucleotide binding to purified F1 from betaR182Q/betaY331W and betaR182K/betaY331W double mutants. It was found that (a) beta-Arg-182 interacts with the gamma-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) beta-Arg-182 has no functional interaction with the beta-phosphate of MgADP or with the magnesium of the magnesium-nucleotide complex in the catalytic sites, and (c) beta-Arg-182 is directly involved in the stabilization of the catalytic transition state. In these features the role of beta-Arg-182 resembles that of another positively charged residue in the catalytic site, the conserved lysine of the Walker A motif, beta-Lys-155. A further role of beta-Arg-182 is suggested, namely involvement in conformational change at the catalytic site beta-alpha subunit interface that is required for multisite catalysis.     

Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly

Combined mutation of "catalytic carboxylates" in both nucleotide binding domains (NBDs) of P-glycoprotein generates a conformation capable of tight binding of 8-azido-ADP (Sauna, Z. E., Müller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry 41, 13989-14000). Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/E1197A bound MgATP and MgADP (1 mol/mol) with K(d) 9.2 and 92 microm, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.     

The (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 has altered ATPase activity after cross-linking alpha and beta subunits at noncatalytic site interfaces

In crystal structures of bovine MF(1), the side chains of alpha F(357) and beta R(372) are near the adenines of nucleotides bound to noncatalytic sites. To determine if during catalysis these side chains must pass through the different arrangements in which they are present in crystal structures, the catalytic properties of the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the TF(1)-ATPase were characterized before and after cross-linking the introduced cysteines with CuCl(2). The unmodified mutant enzyme hydrolyzes MgATP at 50% the rate exhibited by wild type. Detailed comparison of the catalytic properties of the double mutant enzyme before and after cross-linking with those of the wild-type subcomplex revealed the following. Before cross-linking, the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex has less tendency than wild type to release inhibitory MgADP entrapped in a catalytic site during turnover when MgATP binds to noncatalytic sites. Following cross-linking, ATPase activity is reduced 5-fold, and inhibitory MgADP entrapped in a catalytic site during turnover does not release under conditions wherein binding of ATP to noncatalytic sites of the wild-type enzyme promotes release of MgADP from the affected catalytic site. When assayed in the presence of lauryldimethylamine oxide, which prevents turnover-dependent entrapment of inhibitory MgADP in a catalytic site, ATPase activity of the cross-linked form is 47% that of the unmodified mutant enzyme. These results suggest that, during catalysis, the side chains of alpha F(357) and beta R(372) do not pass through the extremely different relative positions in which they exist at the three noncatalytic site interfaces in crystal structures.     

The fluorescence spectrum of the introduced tryptophans in the alpha 3(beta F155W)3gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites.

It has been reported that shifts in the fluorescence emission spectrum of the introduced tryptophans in the betaF155W mutant of Escherichia coli F(1) (bovine heart mitochondria F(1) residue number) can quantitatively distinguish between the number of catalytic sites occupied with ADP and ATP during steady-state ATP hydrolysis (Weber, J., Bowman, C., and Senior, A. E. (1996) J. Biol. Chem. 271, 18711--18718). In contrast, addition of MgADP, Mg-5'-adenylyl beta,gamma-imidophosphate (MgAMP-PNP), and MgATP in 1:1 ratios to the alpha(3)(betaF155W)(3)gamma subcomplex of thermophilic Bacillus PS3 F(1) (TF(1)) induced nearly identical blue shifts in the fluorescence emission maximum that was accompanied by quenching. Addition of 2 mm MgADP induced a slightly greater blue shift and a slight increase in intensity over those observed with 1:1 MgADP. However, addition of 2 mm MgAMP-PNP or MgATP induced a much greater blue shift and substantially enhanced fluorescence intensity over those observed in the presence of stoichiometric MgADP or MgAMP-PNP. It is clear from these results that the fluorescence spectrum of the introduced tryptophans in the betaF155W mutant of TF(1) does not respond in regular increments at any wavelength as catalytic sites are filled with nucleotides. The fluorescence spectrum observed after entrapping MgADP-fluoroaluminate complexes in two catalytic sites of the betaF155W subcomplex indicates that the fluorescence emission spectrum of the enzyme is maximally perturbed when nucleotides are bound to two catalytic sites. This finding is consistent with accumulating evidence suggesting that only two beta subunits in the alpha(3)beta(3)gamma subcomplex of TF(1) can simultaneously exist in the completely closed conformation.     

Tightly bound adenosine diphosphate, which inhibits the activity of mitochondrial F1-ATPase, is located at the catalytic site of the enzyme

The binding of one ADP molecule at the catalytic site of the nucleotide depleted F1-ATPase results in a decrease in the initial rate of ATP hydrolysis. The addition of an equimolar amount of ATP to the nucleotide depleted F1-ATPase leads to the same effect, but, in this case, inhibition is time dependent. The half-time of this process is about 30 s, and the inhibition is correlated with Pi dissociation from the F1-ATPase catalytic site (uni-site catalysis). The F1-ATPase-ADP complex formed under uni-site catalysis conditions can be reactivated in two ways: (i) slow ATP-dependent ADP release from the catalytic site (tau 1/2 20 s) or (ii) binding of Pi in addition to MgADP and the formation of the triple F1-ATPase-MgADP-Pi complex. GTP and GDP are also capable of binding to the catalytic site, however, without changes in the kinetic properties of the F1-ATPase. It is proposed that ATP-dependent dissociation of the F1-ATPase-GDP complex occurs more rapidly, than that of the F1-ATPase-ADP complex.     

Substitution of betaGlu(201) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides

In the crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the side chain oxygen of betaThr(163) interacts directly with Mg(2+) coordinated to 5'-adenylyl beta, gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations. In the unliganded beta subunit present in an open conformation, the hydroxyl of betaThr(163) is hydrogen-bonded to the carboxylate of betaGlu(199). Substitution of betaGlu(201) (equivalent to betaGlu(199) in MF(1)) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 microM ATP. These substitutions lower K(m3) (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively. Fluorescence quenching of alpha(3)(betaE201C/Y341W)(3)gamma and alpha(3)(betaY341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant. These comparisons support the hypothesis that the hydrogen bond observed between the side chains of betaThr(163) and betaGlu(199) in the unliganded catalytic site in the crystal structure of MF(1) stabilizes the open conformation of the catalytic site during ATP hydrolysis.     

Inhibition of yeast mitochondrial F1-ATPase, F0F1-ATPase and submitochondrial particles by rhodamines and ethidium bromide

ATP hydrolysis by F1-ATPase is strongly inhibited by cationic rhodamines; neutral rhodamines are very poor inhibitors. Rhodamine 6G is a noncompetitive inhibitor of purified F0F1-ATPase and submitochondrial particles, however, an uncompetitive inhibitor of F1-ATPase (KI approximately equal to 2.4 microM for all three enzyme forms). Ethidium bromide is a noncompetitive inhibitor of F0F1-ATPase, submitochondrial particles and also F1-ATPase (KI approximately equal to 270 microM). Neither of the inhibitors affects the negative cooperativity (nH approximately equal to 0.7). The non-identical binding sites for rhodamine 6G and ethidium bromide are located on the F1-moiety and are topologically distinct from the catalytic site. Binding of the inhibitors prevents the conformational changes essential for energy transduction. It is concluded that the inhibitor binding sites are involved in proton translocation. In F1-ATPase, binding of MgATP at a catalytic site causes conformational changes, which allosterically induce the correct structure of the rhodamine 6G binding site. In F0F1-ATPase, this conformation of the F1-moiety exists a priori, due to allosteric interactions with F0-subunits. The binding site for ethidium bromide on F1-ATPase does not require substrate binding at the catalytic site and is not affected by F0F1-subunit interactions.     

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.     

Involvement of the "occluded nucleotide conformation" of P-glycoprotein in the catalytic pathway

We found recently that the combined mutation of both "catalytic carboxylate" residues (E552A/E1197A) in mouse P-glycoprotein (Pgp) arrested the protein in an "occluded nucleotide conformation", possibly a stabilized dimer of nucleotide-binding domains (NBDs), that binds MgATP tightly at stoichiometry of 1 mol/mol Pgp [Tombline, G., Bartholomew, L., Urbatsch, I. L., and Senior, A. E. (2004) J. Biol. Chem. 279, 31212-31220]. Here, we further examine this conformation in respect to its potential involvement in the catalytic pathway. The occluded nucleotide conformation is promoted by drugs. Verapamil markedly accelerated the rate of tight binding of MgATP, whereas it did not effect the rate of dissociation. Mutations in "Q-loop" residues that are thought to interfere with communication between drug and catalytic sites prevented the occluded nucleotide conformation, as did covalent reagents N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, which are known to inhibit ATP hydrolysis by reacting in catalytic sites. Mutations of Walker A Ser and Lys residues in combination with E552A/E1197A had the same effect, showing that interaction of these conserved residues with MgATP is required to stabilize the occluded nucleotide conformation. We present an enzymatic scheme that incorporates this conformation. We propose that upon initial loose binding of MgATP at two nucleotide-binding domains (NBDs), together with drug binding, the NBDs dimerize to form the occluded conformation, with one tightly bound MgATP committed to hydrolysis. The pathway progresses such that the tightly bound MgATP enters the transition state and is hydrolyzed. This work suggests that small molecules or peptides that interact at the NBD dimer interface might effectively disable Pgp catalysis.     

Adenine nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli

Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme. Mutant Escherichia coli F(1) that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites. The ESR spectra of the spin-labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme. Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation. Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin-labeled F(1) significantly changes the conformation of the catalytic sites. In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation.     

Adenine nucleotides regulate the functional transition in mitochondrial H+-ATPase and the kinetic behaviour of its ATP-synthetase form

The kinetics of the SMP-catalyzed Pi-ATP exchange and oxidative phosphorylation was studied at variable [MgATP] + + [MgADP] and [MgATP]/[MgADP]. The existence on F1 of a center with a low affinity was demonstrated (KM = 0.4-2.7 mM). Saturation of this center with the Mg2+-complex of one of the nucleotides is obligatory for H+-ATPase to exhibit its ATP synthetase activity. It was found that with a decrease of [MgATP]/[MgADP] the lag periods, tau, of the reactions and KM(Pi) also show a decrease. Besides, in the Pi-ATP exchange reactions delta microH+ (steady-state) diminishes and SMP coupling is enhanced (the Vhydr/Vsynth ratio is decreased). Preincubation of SMP with MgADP eliminates the lags but does not affect the course of the steady-state reaction. It is concluded that F1 when bound to MgATP or MgADP changes to a "more" or "less coupled" conformational state, thus determining the rate of conversion to the ATP-synthetase functional state (ko = tau-1), the threshold potential of this conversion and the kinetic behaviour of ATP-synthetase (KM for Pi).     

The characteristics of the (alpha V371C)3(beta R337C)3 gamma double mutant subcomplex of the TF1-ATPase indicate that the catalytic site at the alpha TP-beta TP interface with bound MgADP in crystal structures of MF1 represents a catalytic site containing inhibitory MgADP

In the MF(1) crystal structure with the MgADP-fluoroaluminate complex bound to two catalytic sites [Menz, R. I., Walker, J. E., and Leslie, A. G. W. (2001) Cell 106, 331-341], the guanidinium of betaR(337) is within 2.9 A of the alpha-oxygen of alphaS(370) and 3.7 A of a methyl group of alphaV(371) at the alpha(E)-beta(HC) interface. To examine the functional role of this contact, the (alphaV(371)C)(3)(betaR(337)C)(3)gamma subcomplex of the TF(1)-ATPase was prepared and characterized. Steady state ATPase activity of the reduced double-mutant is 30% of that of the wild type. Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl(2) cross-linked two alpha-beta pairs, whereas a single alpha-beta pair cross-linked when at least two catalytic sites contained MgADP. The reduced double mutant hydrolyzed substoichiometric ATP 100-fold more rapidly than the enzyme containing two cross-linked alpha-beta pairs. Addition of AlCl(3) and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200 microM MgADP irreversibly inactivated the steady state ATPase activity with rate constants of 1.5 x10(-2) and 4.1 x 10(-2) min(-1), respectively. In contrast, addition of AlCl(3) and NaF to the cross-linked enzyme after incubation with stoichiometric or 200 microM MgADP irreversibly inactivated ATPase activity with a common rate constant of approximately 10(-4) min(-1). Correlation of these results with crystal structures of MF(1) suggests that the catalytic site at the alpha(TP)-beta(TP) interface is loaded first upon addition of nucleotides to nucleotide-depleted F(1)-ATPases and that the catalytic site at the alpha(TP)-beta(TP) interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.     

Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli

The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide. This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain. Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate. Properties of the soluble purified F1-ATPase from each mutant were studied. The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively. 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis. 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site. 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity. 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.