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• W2017383553 abstract "Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F1-ATPase catalytic sites. The mutant enzyme (βF148W) showed normal enzymatic characteristics. The fluorescence responses of β-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP·BeFx complex enhanced the fluorescence at 325 nm. With MgATP, both effects were seen simultaneously. This allowed analysis of bound catalytic site nucleotides directly under steady-state MgATP hydrolysis conditions. At mM concentration of MgATP (Vmax conditions) one of the three catalytic sites was filled with substrate MgATP and the other two sites were filled with product MgADP. A model for F1-ATPase steady-state turnover is presented that encompasses these findings.Given the structural similarity of the P-loop in nucleotide-binding proteins, this approach may prove widely useful. Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F1-ATPase catalytic sites. The mutant enzyme (βF148W) showed normal enzymatic characteristics. The fluorescence responses of β-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP·BeFx complex enhanced the fluorescence at 325 nm. With MgATP, both effects were seen simultaneously. This allowed analysis of bound catalytic site nucleotides directly under steady-state MgATP hydrolysis conditions. At mM concentration of MgATP (Vmax conditions) one of the three catalytic sites was filled with substrate MgATP and the other two sites were filled with product MgADP. A model for F1-ATPase steady-state turnover is presented that encompasses these findings. Given the structural similarity of the P-loop in nucleotide-binding proteins, this approach may prove widely useful. INTRODUCTIONBacteria, mitochondria, and chloroplasts contain a membrane-bound ATP synthase that is responsible for ATP synthesis coupled to oxidation or light capture reactions. In bacteria this enzyme may also act as an ATPase to generate the trans-membrane proton gradient. Understanding the mechanism of ATP synthesis and ATP-driven proton pumping is the goal of many laboratories. While useful hypotheses have been generated (1Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (913) Google Scholar, 2Senior A.E. Physiol. Rev. 1988; 68: 177-231Crossref PubMed Scopus (454) Google Scholar), none is yet widely accepted, because catalysis has not yet been described in sufficient experimental detail.ATP synthases are composed of two sectors, the F0 which forms a trans-membrane proton conduction path, and the F1 which contains three catalytic nucleotide-binding sites, one on each of the three β-subunits. F1 may be isolated in soluble form and has proved valuable for experimental studies. A detailed structure of bovine mitochondrial F1 was recently resolved by x-ray crystallography (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar). Ease of genetic manipulation has meant that the Escherichia coli enzyme has also been extensively studied (for reviews see Refs. 4Fillingame R.H. Krulwich T.A. The Bacteria. Academic Press, New York1990: 345Google Scholar, 5Senior A.E. Annu. Rev. Biophys. Biophys. Chem. 1990; 19: 7-41Crossref PubMed Scopus (328) Google Scholar, 6Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-289Abstract Full Text PDF PubMed Scopus (130) Google Scholar), and because the sequences of the β-subunits are strongly conserved, we may conclude that their structure will be very similar from whatever source.The three catalytic sites of E. coli F1 show widely different affinities for substrate MgATP (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar). Substoichiometric amounts of MgATP bind very tightly to catalytic site one (Kd = 10−10M, Ref. 8Senior A.E. Lee R.S.F. Al-Shawi M.K. Weber J. Arch. Biochem. Biophys. 1992; 297: 340-344Crossref PubMed Scopus (46) Google Scholar). MgATP hydrolysis is very slow when only this first site is occupied (“unisite catalysis”). Because of the low dissociation rates of substrate and products, kinetic and thermodynamic parameters for unisite catalysis can be measured by using the centrifuge column technique to separate enzyme-bound from free ligands (9Grubmeyer C. Cross R.L. Penefsky H.S. J. Biol. Chem. 1982; 257: 12092-12100Abstract Full Text PDF PubMed Google Scholar), and this approach has yielded considerable functional insights into catalysis, particularly when combined with mutational or environmental modification of the catalytic sites (10Senior A.E. Weber J. Al-Shawi M.K. Biochem. Soc. Trans. 1995; 23: 747-752Crossref PubMed Scopus (16) Google Scholar, 11Senior A.E. Al-Shawi M.K. J. Biol. Chem. 1992; 267: 21471-21478Abstract Full Text PDF PubMed Google Scholar, 12Al-Shawi M.K. Senior A.E. Biochemistry. 1992; 31: 878-885Crossref PubMed Scopus (39) Google Scholar, 13Al-Shawi M.K. Senior A.E. Biochemistry. 1992; 31: 886-891Crossref PubMed Scopus (30) Google Scholar).To achieve physiological catalysis rates, however, additional catalytic sites must be filled with MgATP (“multisite catalysis”; for reviews see Refs. 14Penefsky H.S. Cross R.L. Adv. Enzymol. 1991; 64: 173-214PubMed Google Scholar, 15Senior A.E. J. Bioenerg. Biomembr. 1992; 24: 479-484Crossref PubMed Scopus (45) Google Scholar). Analysis of substrate binding parameters under multisite catalysis conditions required the development of a rapid, true equilibrium, analytical technique. Recently, we designed and applied such a technique (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar, 16Weber J. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 1994; 269: 20462-20467Abstract Full Text PDF PubMed Google Scholar, 17Weber J. Senior A.E. J. Biol. Chem. 1996; 271: 3474-3477Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). By specifically introducing a tryptophan residue into the adenine-binding subdomain of E. coli F1 catalytic sites, at position β331, and using its fluorescence to monitor catalytic site occupancy, we were able to show that (i) all three catalytic sites must be filled to obtain physiological MgATP hydrolysis rates; (ii) the rate of hydrolysis of MgATP when only two sites are filled is slow (0-2% of Vmax); and (iii) in absence of Mg2+ (conditions under which there is no hydrolysis) the pronounced binding cooperativity observed with MgATP is absent and all three sites bind ATP with equal affinity.The next logical step in analysis of multisite catalysis is to determine, under steady-state MgATP hydrolysis conditions, what fraction of the three catalytic sites is filled with MgATP and what fraction with MgADP. A prerequisite would be a probe that can differentiate between bound MgATP and bound MgADP. Due to its location in the adenine-binding subdomain of the catalytic sites, residue βTrp-331 responds to binding of the base moiety, and its fluorescence is virtually completely quenched upon addition of MgATP, MgADP, MgAMPPNP, MgTNP-ATP, free ATP, free ADP, or free TNP-ATP (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar, 16Weber J. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 1994; 269: 20462-20467Abstract Full Text PDF PubMed Google Scholar, 17Weber J. Senior A.E. J. Biol. Chem. 1996; 271: 3474-3477Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Thus, no differentiation between bound nucleoside di- or triphosphate is possible on the basis of the βTrp-331 fluorescence signal.Model experiments have suggested that tryptophan fluorescence is susceptible to the electric field of a charge in the immediate environment (18Bivin D.B. Kubota S. Pearlstein R. Morales M.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6791-6795Crossref PubMed Scopus (22) Google Scholar). Thus, a tryptophan residue located close to the γ-phosphate might respond differently to bound MgATP versus MgADP. As shown by comparison of several known protein structures (19Story R.M. Steitz T.A. Nature. 1992; 355: 374-376Crossref PubMed Scopus (558) Google Scholar), and confirmed in the x-ray structure of bovine mitochondrial F1 (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar), the “Homology A” or “P-loop” motif (20Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4220) Google Scholar) surrounds the phosphates of a bound nucleotide molecule. Therefore, to obtain an optical probe with the desired properties, we decided to introduce tryptophan residues close to the P-loop in the catalytic sites of F1.At first glance, none of the amino acid residues within the P-loop in E. coli F1 catalytic sites (β149GGAGVGKT156) appeared to be a promising candidate, i.e. where replacement by Trp would preserve enzyme function. One possible exception was βVal-153, since it was shown that substitution of this Val by Tyr in yeast F1 gave partially functional enzyme (21Shen H. Yao B. Mueller D.M. J. Biol. Chem. 1994; 269: 9424-9428Abstract Full Text PDF PubMed Google Scholar). Other candidates, not within but very close to the P-loop, were βPhe-148 and βAsn-158. The latter is Leu in mitochondrial and chloroplast F1. Therefore, we generated βF148W, βV153W, and βN158W mutants, and to increase the signal-to-noise ratio for fluorescence experiments the mutations were expressed from plasmid pOW1, in which all of the nine natural tryptophan residues present in wild-type F1 have been replaced (22Wilke-Mounts S. Weber J. Grell E. Senior A.E. Arch. Biochem. Biophys. 1994; 309: 363-368Crossref PubMed Scopus (15) Google Scholar). In this paper we describe the effects of the mutations in whole cells and the characteristics of the isolated mutant F1 enzymes, particularly in regard to their fluorescence properties.DISCUSSIONThe first goal of this study was to generate a tryptophan fluorescence probe able to differentiate between an empty catalytic site, an MgATP-filled site, and an MgADP-filled site, in E. coli F1-ATPase. A further goal was to use the probe to establish fractional occupancy of the the three catalytic sites by MgATP and MgADP during steady-state hydrolysis. We introduced Trp in three different positions within or close to the P-loop (20Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4220) Google Scholar) by generating the mutations βF148W, βV153W, and βN158W, and we expressed each mutant in “tryptophan-free F1” (22Wilke-Mounts S. Weber J. Grell E. Senior A.E. Arch. Biochem. Biophys. 1994; 309: 363-368Crossref PubMed Scopus (15) Google Scholar) so that the introduced Trp was the sole Trp residue. Two of the Trp residues, βTrp-153 and βTrp-158, were able to distinguish between an empty and a nucleotide-occupied site but gave the same response (quenching of fluorescence) on binding of MgATP, MgAMPPNP, or MgADP. The third, βTrp-148, fulfilled the requirements, because it gave quenching of fluorescence with MgADP (at 350 nm) and an enhancement of fluorescence with MgAMPPNP and MgADP·BeFx (at 325 nm). Moreover, with MgATP both quenching of fluorescence at 350 nm and an enhancement at 325 nm occurred simultaneously, showing that βTrp-148 differentially senses bound unhydrolyzed MgATP and bound product MgADP in catalytic sites under steady-state hydrolysis conditions. The βF148W mutant strain showed normal growth characteristics under oxidative phosphorylation conditions, and enzymatic characteristics of purified βF148W F1 were similar to those of wild-type enzyme.The fluorescence spectra of residues βTrp-153 and βTrp-158, in absence of nucleotides in the binding site, indicated that their environments are highly nonpolar and that of βTrp-148 indicated a moderately nonpolar environment. Previous work with the βY331W mutant enzyme has shown that in absence of bound nucleotide, the catalytic sites are highly polar and likely to be filled with water molecules (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar). Thus all three Trp residues studied here are probably buried in the protein matrix, pointing away from the nucleotide.Upon binding of MgADP, MgAMPPNP, and MgATP the fluorescence of βTrp-153 and βTrp-158 was quenched. The simplest explanation is that nucleotide binding leads to a change in the protein conformation in the direct environment of either Trp residue. The same explanation can be offered for the quenching of βTrp-148 fluorescence by MgADP. In contrast, binding of MgAMPPNP led to enhancement of βTrp-148 fluorescence, combined with a pronounced blue-shift of the spectrum. The MgADP·BeFx complex (which is known to closely mimic the prehydrolysis MgATP-bound state in myosin, Ref. 37Fisher A.J. Smith C.L. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (634) Google Scholar) gave the same spectrum as MgAMPPNP. This response might also be explained as due to a conformational rearrangement of the protein, albeit different from that observed with MgADP. An attractive alternative explanation, however, is that the electric field of the negative charges introduced with the nucleotide γ-phosphate affects the Trp fluorescence (see Introduction). The fact that the MgADP·BeFx complex caused the same response as MgAMPPNP would be in agreement with either interpretation, as such a complex is isoelectronic and potentially isosteric with MgAMPPNP (and MgATP) (37Fisher A.J. Smith C.L. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (634) Google Scholar).Titration experiments were carried out with βF148W F1 in order to analyze the fluorescence responses with MgADP and MgAMPPNP (Fig. 4, A and B). Quantitative evaluation of the results was affected by the small size of the responses; nevertheless, several conclusions could be drawn with confidence. With either nucleotide, binding cooperativity was evident, and in both cases the data fit reasonably well to a model assuming one site of higher affinity and two sites of lower affinity. This model was established from previous work with the βY331W enzyme (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar), which displays a far superior tryptophan fluorescence signal and is representative of wild-type in enzymatic properties. Thus, for MgADP and MgAMPPNP the lower Kd corresponds to binding to catalytic site one and the higher Kd corresponds to binding to catalytic sites two and three. The fluorescence signals reached saturation at mM ligand concentrations, i.e. 1 order of magnitude above Km(MgATP), as was also seen with the βY331W enzyme (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar), and under these conditions all three catalytic sites would therefore be filled with ligand.We next studied the fluorescence responses of βTrp-148 upon titration of βF148W F1 with MgATP concentrations ranging from 0.2 µM up to mM. The data were described in Fig. 5, A-C, and show several important features. At MgATP concentrations sufficient to achieve Vmax rates of hydrolysis, all three catalytic sites became occupied by nucleotide, consistent with our previous work (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar). At the lowest concentration of MgATP of 0.2 µM (where [MgATP] ≈ [F1]) around 0.6 mol total nucleotide was bound per mol F1, corresponding to binding to the highest affinity catalytic site one. When the concentration of MgATP in the medium was raised to 5 µM and above, the average nucleotide occupancy rose, as catalytic site two began to fill. However, even immediately after mixing the amount of bound MgATP never exceeded 1 mol/mol, which indicates that upon binding of MgATP to site two, hydrolysis of MgATP bound at site one was a fast reaction, and release of MgADP from site one was slow, consistent with the slow net turnover rate under these conditions. Further increase of the MgATP concentration up to mM concentrations was sufficient to fill all three catalytic sites and to achieve Vmax rates of hydrolysis. Under steady-state turnover conditions one-third of the catalytic sites contained MgATP and two-thirds contained MgADP (Fig. 6).A model for steady-state, “multisite” MgATP hydrolysis based on these findings is presented in Fig. 7. In the model, the catalytic site with highest affinity for MgATP is designated as H, the site with intermediate affinity is designated as M, and the site with lowest affinity for MgATP is designated as L. After release of MgADP from site L in the previous reaction cycle, site H contains MgATP, site M contains MgADP, and site L is transiently unoccupied (State D in Fig. 7). Then site L fills rapidly with MgATP from the medium (kcat/KM ~106M−1 s−1) to yield State A in Fig. 7. Instantaneously, MgATP at site H is hydrolyzed to MgADP + Pi; this process triggers a synchronized switch in affinity at all three catalytic sites, which is shown by the arrows in State B, Fig. 7. In another fast reaction, Pi is released from the (now) site M, resulting in State C, Fig. 7. Subsequent MgADP release from site L is the rate-limiting step in the cycle. Consequently, State C is the most populous state of all the enzyme molecules present under steady-state MgATP hydrolysis conditions, and the fluorescence response obtained reflects State C. The model incorporates information from previous models in that it involves three catalytic sites, of widely differing affinity for MgATP, with positive catalytic cooperativity observed on binding of MgATP to the lowest affinity site, and a synchronized change in affinity of the sites at one step of the catalytic cycle (1Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (913) Google Scholar, 2Senior A.E. Physiol. Rev. 1988; 68: 177-231Crossref PubMed Scopus (454) Google Scholar). The essential feature of this model is that it accounts for the new finding from the fluorescence experiments that two of the sites are occupied by MgADP and only one by MgATP in the form of enzyme that predominates in time-average during the catalytic cycle in steady-state MgATP hydrolysis. It may be inferred that this is a necessary feature for the reverse reaction, namely steady-state Δ˜µH+-driven MgATP synthesis.In the structure of mitochondrial F1 obtained by x-ray crystallography (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar), one catalytic site is occupied by MgAMPPNP and one site by MgADP, whereas the other site is empty. In our model (Fig. 7) this corresponds to State D. Our data show that this form of the enzyme is present as only a small fraction of the molecules during steady-state catalysis; nevertheless, it is not unreasonable to suppose that under the crystallization conditions used (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar) this form of the enzyme may have been selectively sequestered into the crystals.It is relevant to point out that during catalysis there are potentially at least two states through which the catalytic site passes in addition to the MgATP- and the MgADP-bound states, namely the catalytic transition state and the “MgADP + Pi”-bound state. X-ray crystallographic analysis of other ATP- or GTP-hydrolyzing enzymes has suggested that the catalytic transition state is mimicked by bound MgADP·AlF−4 complex (37Fisher A.J. Smith C.L. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (634) Google Scholar, 39Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (530) Google Scholar). We generated the βF148W F1·MgADP·AlF−4 complex (see “Experimental Procedures”) and found that its fluorescence spectrum resembled that seen on addition of mM concentration of MgATP (as in Fig. 3C), that is to say it contained characteristics of both MgADP-bound and MgAMPPNP-bound forms. One interpretation of this result is that this spectrum truly represents that of the catalytic transition state. However, the experiment is ambiguous because it is also possible that under these conditions a fraction of the catalytic sites is in the MgADP-bound state. This would be the case, for example, if the F1 mechanism mandates that only one of the catalytic sites can be in the catalytic transition state at any given time, as appears to be the case for P-glycoprotein (41Senior A.E. Al-Shawi M.K. Urbatsch I.L. FEBS Lett. 1996; 377: 285-289Google Scholar). Since the time spent in the catalytic transition state is very short in comparison to the time spent in the ground states, the contribution to the overall fluorescence signal during steady-state catalysis should be small. It would be valuable to obtain a probe that sensed the transition state specifically.We also attempted to determine the contribution of the “MgADP + Pi”-bound state to the overall fluorescence signal. It was found that addition of Pi together with MgADP had no effect on the βTrp-148 fluorescence response (see “Results”). In all likelihood, however, this is due to the fact that an F1·MgADP + Pi complex was not generated. Previous work has shown that Pi by itself has very weak binding affinity at catalytic sites in soluble F1, and addition of Pi does not affect the binding affinity of MgADP (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar, 42Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 12653-12658Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Therefore, there are limitations as to how far we can dissect the catalytic cycle in respect to differential rates of release of Pi versus MgADP using the current data. However, it should be pointed out that the model of Fig. 7 must be essentially correct in that, since a state with all three catalytic sites occupied with nucleotide (one MgATP and two MgADP) is the predominant long-lived species during the catalytic cycle, there is clearly no possibility that release of MgADP precedes that of Pi.Given the small size of the underlying fluorescence responses and other considerations discussed above, we stress that the model of Fig. 7 is a working hypothesis. It is hoped that in the future different Trp probes can be developed to test and extend the model, for example by specifically reporting the catalytic transition state or the presence or absence of Pi. The most important contribution of the current work is to demonstrate for the first time that reaction events occurring at the catalytic sites of F1-ATPase during steady-state catalysis are amenable to analysis using direct optical probes.Finally it may be noted that, given the general similarity of structure of the P-loop sequence in many nucleotide-binding proteins, and the fact that βTrp-148 was specifically inserted in the position immediately preceding the first residue of the P-loop, there seems to be a good chance that insertion of Trp at the equivalent position might provide a valuable probe to distinguish bound nucleoside triphosphate from diphosphate in a range of different proteins and enzymes. INTRODUCTIONBacteria, mitochondria, and chloroplasts contain a membrane-bound ATP synthase that is responsible for ATP synthesis coupled to oxidation or light capture reactions. In bacteria this enzyme may also act as an ATPase to generate the trans-membrane proton gradient. Understanding the mechanism of ATP synthesis and ATP-driven proton pumping is the goal of many laboratories. While useful hypotheses have been generated (1Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (913) Google Scholar, 2Senior A.E. Physiol. Rev. 1988; 68: 177-231Crossref PubMed Scopus (454) Google Scholar), none is yet widely accepted, because catalysis has not yet been described in sufficient experimental detail.ATP synthases are composed of two sectors, the F0 which forms a trans-membrane proton conduction path, and the F1 which contains three catalytic nucleotide-binding sites, one on each of the three β-subunits. F1 may be isolated in soluble form and has proved valuable for experimental studies. A detailed structure of bovine mitochondrial F1 was recently resolved by x-ray crystallography (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2734) Google Scholar). Ease of genetic manipulation has meant that the Escherichia coli enzyme has also been extensively studied (for reviews see Refs. 4Fillingame R.H. Krulwich T.A. The Bacteria. Academic Press, New York1990: 345Google Scholar, 5Senior A.E. Annu. Rev. Biophys. Biophys. Chem. 1990; 19: 7-41Crossref PubMed Scopus (328) Google Scholar, 6Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-289Abstract Full Text PDF PubMed Scopus (130) Google Scholar), and because the sequences of the β-subunits are strongly conserved, we may conclude that their structure will be very similar from whatever source.The three catalytic sites of E. coli F1 show widely different affinities for substrate MgATP (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar). Substoichiometric amounts of MgATP bind very tightly to catalytic site one (Kd = 10−10M, Ref. 8Senior A.E. Lee R.S.F. Al-Shawi M.K. Weber J. Arch. Biochem. Biophys. 1992; 297: 340-344Crossref PubMed Scopus (46) Google Scholar). MgATP hydrolysis is very slow when only this first site is occupied (“unisite catalysis”). Because of the low dissociation rates of substrate and products, kinetic and thermodynamic parameters for unisite catalysis can be measured by using the centrifuge column technique to separate enzyme-bound from free ligands (9Grubmeyer C. Cross R.L. Penefsky H.S. J. Biol. Chem. 1982; 257: 12092-12100Abstract Full Text PDF PubMed Google Scholar), and this approach has yielded considerable functional insights into catalysis, particularly when combined with mutational or environmental modification of the catalytic sites (10Senior A.E. Weber J. Al-Shawi M.K. Biochem. Soc. Trans. 1995; 23: 747-752Crossref PubMed Scopus (16) Google Scholar, 11Senior A.E. Al-Shawi M.K. J. Biol. Chem. 1992; 267: 21471-21478Abstract Full Text PDF PubMed Google Scholar, 12Al-Shawi M.K. Senior A.E. Biochemistry. 1992; 31: 878-885Crossref PubMed Scopus (39) Google Scholar, 13Al-Shawi M.K. Senior A.E. Biochemistry. 1992; 31: 886-891Crossref PubMed Scopus (30) Google Scholar).To achieve physiological catalysis rates, however, additional catalytic sites must be filled with MgATP (“multisite catalysis”; for reviews see Refs. 14Penefsky H.S. Cross R.L. Adv. Enzymol. 1991; 64: 173-214PubMed Google Scholar, 15Senior A.E. J. Bioenerg. Biomembr. 1992; 24: 479-484Crossref PubMed Scopus (45) Google Scholar). Analysis of substrate binding parameters under multisite catalysis conditions required the development of a rapid, true equilibrium, analytical technique. Recently, we designed and applied such a technique (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar, 16Weber J. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 1994; 269: 20462-20467Abstract Full Text PDF PubMed Google Scholar, 17Weber J. Senior A.E. J. Biol. Chem. 1996; 271: 3474-3477Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). By specifically introducing a tryptophan residue into the adenine-binding subdomain of E. coli F1 catalytic sites, at position β331, and using its fluorescence to monitor catalytic site occupancy, we were able to show that (i) all three catalytic sites must be filled to obtain physiological MgATP hydrolysis rates; (ii) the rate of hydrolysis of MgATP when only two sites are filled is slow (0-2% of Vmax); and (iii) in absence of Mg2+ (conditions under which there is no hydrolysis) the pronounced binding cooperativity observed with MgATP is absent and all three sites bind ATP with equal affinity.The next logical step in analysis of multisite catalysis is to determine, under steady-state MgATP hydrolysis conditions, what fraction of the three catalytic sites is filled with MgATP and what fraction with MgADP. A prerequisite would be a probe that can differentiate between bound MgATP and bound MgADP. Due to its location in the adenine-binding subdomain of the catalytic sites, residue βTrp-331 responds to binding of the base moiety, and its fluorescence is virtually completely quenched upon addition of MgATP, MgADP, MgAMPPNP, MgTNP-ATP, free ATP, free ADP, or free TNP-ATP (7Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar, 16Weber J. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 1994; 269: 20462-20467Abstract Full Text PDF PubMed Google Scholar, 17Weber J. Senior A.E. J. Biol. Chem. 1996; 271: 3474-3477Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Thus, no differentiation be" @default.
• W2017383553 created "2016-06-24" @default.
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• W2017383553 date "1996-08-01" @default.
• W2017383553 modified "2023-10-02" @default.
• W2017383553 title "Specific Tryptophan Substitution in Catalytic Sites of Escherichia coli F1-ATPase Allows Differentiation between Bound Substrate ATP and Product ADP in Steady-state Catalysis" @default.
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