Bi-site catalysis in F1-ATPase: does it exist?

The mechanism of action of F(1)F(0)-ATP synthase is controversial. Some favor a tri-site mechanism, where substrate must fill all three catalytic sites for activity, others a bi-site mechanism, where one of the three sites is always unoccupied. New approaches were applied to examine this question. First, ITP was used as hydrolysis substrate; lower binding affinities of ITP versus ATP enable more accurate assessment of sites occupancy. Second, distributions of all eight possible enzyme species (with zero, one, two or three sites filled) as fraction of total enzyme population at each ITP concentration were calculated, and compared with measured ITPase activity. Confirming data were obtained with ATP as substrate. Third, we performed a theoretical analysis of possible bi-site mechanisms. The results argue convincingly that bi-site hydrolysis activity is negligible, and may not even exist. Effectively, tri-site hydrolysis is the only mechanism. We argue that only tri-site hydrolysis drives subunit rotation. Theoretical analyses of possible bi-site mechanisms reveal serious flaws, not previously recognized. One is that, in bi-site catalysis, the predicted direction of subunit rotation is the same for both ATP synthesis and hydrolysis; a second is that infrequently occurring enzyme species are required.

Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells.

P-glycoprotein confers multidrug resistance in mammalian cells and basic structure-function studies of it are germane to anti-cancer and anti-AIDS therapy. Pure, detergent-soluble mouse MDR3 and human MDR1 P-glycoproteins have recently been obtained in sufficient quantity for high-resolution structure analysis after expression in Pichia pastoris (N. Lerner-Marmarosh et al. (1999) J. Biol. Chem. 274, 34711-34718). The degree of glycosylation of these preparations was unknown, and was of relevance for crystallization studies. Therefore mutant proteins in which the N-glycosylation sites were eliminated (Asn --> Gln in mouse MDR3 Pgp, Asn --> Gln or Ala in human MDR1 Pgp) were expressed in P. pastoris and purified to homogeneity. Yields of mutant Pgp were the same as for parent wild-type proteins. Nucleotide-binding and catalytic (ATPase) characteristics were completely normal in the mutant proteins. Mass spectrometry indicated that mutant and wild-type proteins did not differ significantly in mass, demonstrating that the wild-type proteins contain no N-glycosylation.

Cysteines 431 and 1074 are responsible for inhibitory disulfide cross-linking between the two nucleotide-binding sites in human P-glycoprotein.

Human wild-type and Cys-less P-glycoproteins were expressed in Pichia pastoris and purified in high yield in detergent-soluble form. Both ran on SDS gels as a single 140-kDa band in the presence of reducing agent and showed strong verapamil-stimulated ATPase activity in the presence of added lipid. The wild type showed spontaneous formation of higher molecular mass species in the absence of reducing agent, and its ATPase was activated by dithiothreitol. Oxidation with Cu(2+) generated the same higher molecular mass species, primarily at 200 and approximately 300 kDa, in high yield. Cross-linking was reversed by dithiothreitol and prevented by pretreatment with N-ethylmaleimide. Using proteins containing different combinations of naturally occurring Cys residues, it was demonstrated that an inhibitory intramolecular disulfide bond forms between Cys-431 and Cys-1074 (located in the Walker A sequences of nucleotide-binding sites 1 and 2, respectively), giving rise to the 200-kDa species. In addition, dimeric P-glycoprotein species ( approximately 300 kDa) form by intermolecular disulfide bonding between Cys-431 and Cys-1074. The ready formation of the intramolecular disulfide between Cys-431 and Cys-1074 establishes that the two nucleotide-binding sites of P-glycoprotein are structurally very close and capable of intimate functional interaction, consistent with available information on the catalytic mechanism. Formation of such a disulfide in vivo could, in principle, underlie a regulatory mechanism and might provide a means of intervention to inhibit P-glycoprotein.

ATP-driven rotation of the gamma subunit in F(1)-ATPase.

We present a mechanism for F(1)-ATPase in which hydrolysis of MgATP in the high-affinity catalytic site at the alpha/beta interface drives rotation of the gamma subunit via conformational changes in the alpha subunit. During hydrolysis, transition state formation and separation of P(i) from MgADP causes movement of portions of alpha, transmitted via two Arg residues which are hydrogen-bonded to the gamma-phosphate of MgATP, alphaArg376 and betaArg182; the latter is also hydrogen-bonded to interfacial alpha residues between alpha346 and alpha349. Changes in alpha conformation then push on gamma, resulting in rotation. Supporting evidence from the literature and from new data is discussed.

Investigation of the role of glutamine-471 and glutamine-1114 in the two catalytic sites of P-glycoprotein.

P-glycoprotein, also known as multidrug resistance protein, pumps drugs out of cells using ATP hydrolysis as the energy source. Glutamine-471 and the corresponding glutamine-1114 in the two catalytic sites of P-glycoprotein are conserved in ABC transporters. X-ray structures show that they lie close to the bound nucleotide. Proposed functional roles are (1) activation of the attacking water for ATP hydrolysis, (2) coordination of the essential Mg(2+) cofactor in Mg nucleotide, and (3) signal communication between catalytic site reaction chemistry and drug-binding sites. We made mutations Q471A, Q471E, Q1114A, and Q1114E in mouse MDR3 P-glycoprotein. Pure mutant and wild-type proteins were prepared and subjected to enzymatic and biochemical characterization. We conclude from the results that the primary role of this glutamine residue is in interdomain signal communication. Coordination of the Mg(2+) cofactor is not a critical functional role, neither is activation of the attacking water molecule, although an auxiliary role in positioning the water cannot be ruled out. We found that equivalent mutations (Ala or Glu) in either of the two P-glycoprotein catalytic sites produced the same effects, implying functional symmetry of the two sites.

New probes of the F1-ATPase catalytic transition state reveal that two of the three catalytic sites can assume a transition state conformation simultaneously.

MgADP in combination with fluoroscandium (ScFx) is shown to form a potently inhibitory, tightly bound, noncovalent complex at the catalytic sites of F(1)-ATPase. The F(1).MgADP.ScFx complex mimics a catalytic transition state. Notably, ScFx caused large enhancement of MgADP binding affinity at both catalytic sites 1 and 2, with little effect at site 3. These results indicate that sites 1 and 2 may form a transition state conformation. A new direct optical probe of F(1)-ATPase catalytic transition state conformation is also reported, namely, substantial enhancement of fluorescence emission of residue beta-Trp-148 observed upon binding of MgADP.ScFx or MgIDP. ScFx. Using this fluorescence signal, titrations were performed with MgIDP.ScFx which demonstrated that catalytic sites 1 and 2 can both form a transition state conformation but site 3 cannot. Supporting data were obtained using MgIDP-fluoroaluminate. Current models of the MgATP hydrolysis mechanism uniformly make the assumption that only one catalytic site hydrolyzes MgATP at any one time. The fluorometal analogues demonstrate that two sites have the capability to form the transition state simultaneously.

Functional analysis of a tryptophan-less P-glycoprotein: a tool for tryptophan insertion and fluorescence spectroscopy.

P-glycoprotein (Pgp) functions as an ATP-dependent drug efflux pump to confer multidrug resistance to tumor cells. In the absence of a high-resolution structure for this protein, several important and intriguing aspects of Pgp structure and function remain poorly understood. Fluorescence spectroscopy of endogenous or genetically engineered tryptophan residues represents a potentially powerful method to probe static and dynamic aspects of Pgp at high resolution. We have used site-directed mutagenesis to modify the wild-type (WT) mouse mdr3 Pgp for tryptophan fluorescence spectroscopy by replacement of all 11 tryptophan residues individually with phenylalanine. None of the 11 tryptophans were found to be absolutely essential for Pgp activity, because Chinese hamster ovary cells transfected and overexpressing this mutant Trp-less mdr3 cDNA (mdr3F(1-11)) become multidrug-resistant and can carry out active transport of vinblastine, colchicine, and Calcein-AM. The mdr3F(1-11) mutant has reduced activity compared with WT Mdr3, and shows a unique pattern of drug resistance clearly distinct from WT and, as opposed to the latter, can neither confer FK-506 resistance nor functionally complement ste6 in yeast. Studies with Pgp mutants containing either single or double tryptophan residues or with chimeric molecules constructed between wild-type Pgp and mdr3F(1-11) indicated that no single tryptophan residue was responsible for the reduced activity of the mdr3F(1-11) mutant. Likewise, all but one chimeric Pgp preserved the unique drug resistance profile of the mdr3F(1-11) mutant. Altogether, we show that a Trp-less Pgp is functionally active and can be used as a molecular backbone for insertion of tryptophans in strategic locations to probe various aspects of Pgp function.

ATP synthase: what we know about ATP hydrolysis and what we do not know about ATP synthesis.

In ATP synthase, X-ray structures, demonstration of ATP-driven gamma-subunit rotation, and tryptophan fluorescence techniques to determine catalytic site occupancy and nucleotide binding affinities have resulted in pronounced progress in understanding ATP hydrolysis, for which a mechanism is presented here. In contrast, ATP synthesis remains enigmatic. The molecular mechanism by which ADP is bound in presence of a high ATP/ADP concentration ratio is a fundamental unknown; similarly P(i) binding is not understood. Techniques to measure catalytic site occupancy and ligand binding affinity changes during net ATP synthesis are much needed. Relation of these parameters to gamma-rotation is a further goal. A speculative model for ATP synthesis is offered.

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.

Features of F(1)-ATPase catalytic and noncatalytic sites revealed by fluorescence lifetimes and acrylamide quenching of specifically inserted tryptophan residues.

Catalytic and noncatalytic nucleotide sites of the F(1) sector of ATP synthase were characterized by tryptophan fluorescence techniques. Seven Trp residues inserted in varied microenvironments in the catalytic sites, and one in the noncatalytic sites, were studied in mutant F(1) enzymes which were otherwise devoid of Trp. Parameters measured were fluorescence lifetimes and dynamic and static quenching by acrylamide in the absence or presence of nucleotide. The results indicated that the solution structures of the mutant enzymes were consistent with reported crystal structures. In enzyme with three empty noncatalytic sites, all sites were relatively inaccessible to acrylamide, indicating a closed conformation. In contrast, when the three catalytic sites were empty, they were relatively and equally accessible to acrylamide, indicating an open conformation. This was the case in the presence or absence of Mg(2+). Residue beta-Trp-331 has been extensively used previously to determine nucleotide binding parameters in F(1). Results here showed that in betaY331W mutant F(1), each of the three beta-Trp-331 residues has an unusually long fluorescence lifetime, confirming that each contributes equally to the overall fluorescence signal.

The catalytic transition state in ATP synthase.

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP-fluoroaluminate, Mg-ADP-fluoroscandium, and corresponding Mg-IDP-fluorometals as transition-state analogs; (2) fluorescence signals of beta-Trp331 and beta-Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of alpha-Arg376, acting as an "arginine finger" residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site alpha/beta subunit-interface conformational movement, which is linked to gamma-subunit rotation.

Rate acceleration of ATP hydrolysis by F(1)F(o)-ATP synthase.

The rate acceleration of ATP hydrolysis by F(1)F(o)-ATP synthase is of the order of 10(11)-fold. We present a cyclic enzyme mechanism for the reaction, relate it to known F(1) X-ray structure and speculate on the linkage between enzyme reaction intermediates and subunit rotation. Next, we describe five factors known to be important in the Escherichia coli enzyme for the rate acceleration. First, the provision of substrate binding energy by residues lining the catalytic site is substantial; beta-Lys155 and beta-Arg182 are specific examples, both of which differentially support substrate MgATP versus product MgADP binding. Second, octahedral coordination of the Mg(2+) in MgATP is crucial for both catalysis and catalytic site asymmetry. The residues involved are beta-Thr156, beta-Glu185 and beta-Asp242. Third, there is stabilization of a pentacoordinate phosphorus catalytic transition state by residues beta-Lys155, beta-Arg182 and alpha-Arg376. Fourth, residue beta-Glu181 binds the substrate water and stabilizes the catalytic transition state. Fifth, there is strong positive catalytic cooperativity, with binding of MgATP at all three sites yielding the maximum rate (V(max)); the molecular basis of this factor remains to be elucidated.

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.

Large scale purification of detergent-soluble P-glycoprotein from Pichia pastoris cells and characterization of nucleotide binding properties of wild-type, Walker A, and Walker B mutant proteins.

P-glycoprotein (Pgp; mouse MDR3) was expressed in Pichia pastoris, grown in fermentor culture, and purified. The final pure product is of high specific ATPase activity and is soluble at low detergent concentration. 120 g of cells yielded 6 mg of pure Pgp; >4 kg of cells were obtained from a single fermentor run. Properties of the pure protein were similar to those of previous preparations, except there was significant ATPase activity in absence of added lipid. Mutant mouse MDR3 P-glycoproteins were purified by the same procedure after growth of cells in flask culture, with similar yields and purity. This procedure should open up new avenues of structural, biophysical, and biochemical studies of Pgp. Equilibrium nucleotide-binding parameters of wild-type mouse MDR3 Pgp were studied using 2'-(3')-O-(2,4,6-trinitrophenyl)adenosine tri- and diphosphate. Both analogs were found to bind with K(d) in the low micromolar range, to a single class of site, with no evidence of cooperativity. ATP displacement of the analogs was seen. Similar binding was seen with K429R/K1072R and D551N/D1196N mutant mouse MDR3 Pgp, showing that these Walker A and B mutations had no significant effect on affinity or stoichiometry of nucleotide binding. These residues, known to be critical for catalysis, are concluded to be involved primarily in stabilization of the catalytic transition state in Pgp.

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.

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.

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.

Effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three F1-ATPase catalytic sites measured using specific tryptophan probes.

Equilibrium nucleotide binding to the three catalytic sites of Escherichia coli F1-ATPase was measured in the presence of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin to elucidate mechanisms of inhibition. Fluorescence signals of beta-Trp-331 and beta-Trp-148 substituted in catalytic sites were used to determine nucleotide binding parameters. Azide brought about small decreases in Kd(MgATP) and Kd(MgADP). Notably, under MgATP hydrolysis conditions, it caused all enzyme molecules to assume a state with three catalytic site-bound MgATP and zero bound MgADP. These results rule out the idea that azide inhibits by "trapping" MgADP. Rather, azide blocks the step at which signal transmission between catalytic sites promotes multisite hydrolysis. Aurovertin bound with stoichiometry of 1.8 (mol/mol of F1) and allowed significant residual turnover. Cycling of the aurovertin-free beta-subunit catalytic site through three normal conformations was indicated by MgATP binding data. Aurovertin did not change the normal ratio of 1 bound MgATP/2 bound MgADP in catalytic sites. The results indicate that it acts to slow the switch of catalytic site affinities ("binding change step") subsequent to MgATP hydrolysis. Dicyclohexylcarbodiimide shifted the ratio of catalytic site-bound MgATP/MgADP from 1:2 to 1.6:1.4, without affecting Kd(MgATP) values. Like azide, it also appears to affect activity at the step after MgATP binding, in which signal transmission between catalytic sites promotes MgATP hydrolysis.

Catalytic mechanism of P-glycoprotein.

We generated Chinese hamster ovary cells which are highly multidrug-resistant by selection in colchicine. Purified plasma membranes from these cells are enriched in P-glycoprotein (Pgp), up to 32% w/w of membrane protein. From plasma membranes we purified Pgp to homogeneity and reconstituted it in proteoliposomes. Both plasma membranes and purified reconstituted Pgp show drug-stimulated ATPase activity (approximately 20 s-1), comparable to other transport ATPases. These materials enable investigation and characterization of the catalytic sites and mechanism. Various approaches have been used, notably enzyme kinetics, photoaffinity and other covalent labelling, use of vanadate as transition-state analog, and inhibition by beryllium and aluminum fluoride. Both Pgp nucleotide sites hydrolyse MgATP and are of relatively low specificity and affinity for nucleotides. Trapping of nucleotide by vanadate in either site blocks catalysis at both sites; covalent inactivation of either site completely blocks turnover. Therefore the catalytic sites interact strongly, and it appears that when one site enters the transition-state conformation the other site is prohibited from doing so. A minimal reaction scheme for ATP hydrolysis has been determined. We have proposed an alternating catalytic sites scheme, in which drug-transport is coupled to relaxation of a high chemical potential conformation of the catalytic site (Pgp.MgADP.Pi) which is generated by the hydrolysis step itself. Photoaffinity labelling of Pgp catalytic sites has revealed equivalent Tyr residues which lie close to the adenine ring of bound MgATP in both sites.

Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase.

Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity. Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides. In contrast, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-state, Vmax MgATP hydrolysis.