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• W2008875470 abstract "Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the “DEVD” Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018–2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s–1versus 27 s–1, respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439–17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis. Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the “DEVD” Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018–2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s–1versus 27 s–1, respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439–17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis. SecA is an essential component of the protein translocation machinery in prokaryotes whose function is to transport preproteins across the cytoplasmic membrane. According to current model mechanisms for protein translocation, SecA takes up a precursor of secretory protein from the chaperone SecB, or from the cytosol, and inserts it through a membrane translocon formed by SecYEG and SecDFyajC proteins via repeated cycles of membrane insertion and retraction associated with pre-protein binding and release. SecA possesses an ATPase activity that is essential for protein transport (see reviews Refs. 1de Keyzer J. van der Does C. Driessen A.J. Cell Mol. Life Sci. 2003; 60: 2034-2052Crossref PubMed Scopus (162) Google Scholar, 2Mori H. Ito K. Trends Microbiol. 2001; 9: 494-500Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 3Economou A. Mol. Membr. Biol. 2002; 19: 159-169Crossref PubMed Scopus (40) Google Scholar). The coupling between SecA-catalyzed ATP binding, hydrolysis, and product dissociation events, and the translocon and preprotein binding and release events underlies the mechanics of the protein transport process. SecA has a high-affinity ATP-binding site that comprises Walker A and B motifs responsible for coordinating the α- and β-phosphates of the nucleotide and the Mg2+ ion, respectively (Fig. 1) (4Geourjon C. Orelle C. Steinfels E. Blanchet C. Deleage G. Di Pietro A. Jault J.M. Trends Biochem. Sci. 2001; 26: 539-544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). There also appears to be a low-affinity nucleotide-binding site on SecA, although it is not known whether this site is catalytically active and how it might contribute to the protein translocation mechanism (5Fak J.J. Itkin A. Ciobanu D.D. Lin E.C. Song X.J. Chou Y.T. Gierasch L.M. Hunt J.F. Biochemistry. 2004; 43: 7307-7327Crossref PubMed Scopus (42) Google Scholar, 6Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar, 7van der Wolk J.P. Boorsma A. Knoche M. Schafer H.J. Driessen A.J. Biochemistry. 1997; 36: 14924-14929Crossref PubMed Scopus (13) Google Scholar, 8den Blaauwen T. Fekkes P. de Wit J.G. Kuiper W. Driessen A.J. Biochemistry. 1996; 35: 11994-12004Crossref PubMed Scopus (67) Google Scholar, 9van der Wolk J.P. Klose M. de Wit J.G. den Blaauwen T. Freudl R. Driessen A.J. J. Biol. Chem. 1995; 270: 18975-18982Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 10Miller A. Wang L. Kendall D.A. Biochemistry. 2002; 41: 5325-5332Crossref PubMed Scopus (23) Google Scholar). The high-affinity nucleotide-binding site is better defined, but even its exact role in protein translocation is not completely clear (5Fak J.J. Itkin A. Ciobanu D.D. Lin E.C. Song X.J. Chou Y.T. Gierasch L.M. Hunt J.F. Biochemistry. 2004; 43: 7307-7327Crossref PubMed Scopus (42) Google Scholar, 11Economou A. Pogliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 12Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar, 13van der Wolk J. Klose M. Breukink E. Demel R.A. de Kruijff B. Freudl R. Driessen A.J. Mol. Microbiol. 1993; 8: 31-42Crossref PubMed Scopus (60) Google Scholar, 14de Keyzer J. van der Does C. Kloosterman T.G. Driessen A.J. J. Biol. Chem. 2003; 278: 29581-29586Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 15Rajapandi T. Oliver D. Mol. Microbiol. 1996; 20: 43-51Crossref PubMed Scopus (33) Google Scholar). SecA has a low endogenous ATPase activity that is stimulated 5–10-fold by the presence of SecYEG translocon/membrane and pre-protein, highlighting the link between the ATPase and protein translocation activities (16Natale P. Swaving J. van der Does C. de Keyzer J. Driessen A.J. J. Biol. Chem. 2004; 279: 13769-13777Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 17Sianidis G. Karamanou S. Vrontou E. Boulias K. Repanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 2001; 20: 961-970Crossref PubMed Scopus (95) Google Scholar, 18Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D. Wickner W. EMBO J. 1989; 8: 961-966Crossref PubMed Scopus (310) Google Scholar). Studies probing this link have shown that binding of AMP-PNP 1The abbreviations used are: AMP-PNP, adenosine 5′-(β,γ-imino)-triphosphate; ATPγS, adenosine 5′-3-O-(thio)triphosphate; PBP, phosphate-binding protein; MDCC, 7-diethylamino-3-((((2-maleimidyl)ethyl)-amino)carbonyl)coumarin. (a non-hydrolyzable ATP analog) to the high-affinity site stabilizes the interaction between SecA and SecYEG/membrane (11Economou A. Pogliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 14de Keyzer J. van der Does C. Kloosterman T.G. Driessen A.J. J. Biol. Chem. 2003; 278: 29581-29586Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 19Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (483) Google Scholar, 20Eichler J. Wickner W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5574-5581Crossref PubMed Scopus (73) Google Scholar, 21van der Does C. Manting E.H. Kaufmann A. Lutz M. Driessen A.J. Biochemistry. 1998; 37: 201-210Crossref PubMed Scopus (94) Google Scholar, 22Zito C.R. Oliver D. J. Biol. Chem. 2003; 278: 40640-40646Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). One interpretation of this result is that ATP binding to SecA triggers its insertion into the translocon. However, because the interaction of AMP-PNP with SecA is ∼1000-fold weaker than that of ATP, it is not entirely clear whether this nucleotide analog can accurately mimic the effects of ATP binding to SecA (5Fak J.J. Itkin A. Ciobanu D.D. Lin E.C. Song X.J. Chou Y.T. Gierasch L.M. Hunt J.F. Biochemistry. 2004; 43: 7307-7327Crossref PubMed Scopus (42) Google Scholar). Thus, the question of what role ATP binding to SecA plays in the protein translocation mechanism remains open. Another notable finding is that ADP binding to the high-affinity site stabilizes a compact conformation of SecA (ground state) that has low affinity for the SecYEG/membrane (5Fak J.J. Itkin A. Ciobanu D.D. Lin E.C. Song X.J. Chou Y.T. Gierasch L.M. Hunt J.F. Biochemistry. 2004; 43: 7307-7327Crossref PubMed Scopus (42) Google Scholar, 8den Blaauwen T. Fekkes P. de Wit J.G. Kuiper W. Driessen A.J. Biochemistry. 1996; 35: 11994-12004Crossref PubMed Scopus (67) Google Scholar, 12Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar, 23Ulbrandt N.D. London E. Oliver D.B. J. Biol. Chem. 1992; 267: 15184-15192Abstract Full Text PDF PubMed Google Scholar). This result suggests that following ATP hydrolysis, the ADP-bound SecA undergoes retraction from the translocon to complete one reaction cycle. However, the apo (nucleotide free) form of SecA can also exist in a compact conformation with low affinity for the translocon (5Fak J.J. Itkin A. Ciobanu D.D. Lin E.C. Song X.J. Chou Y.T. Gierasch L.M. Hunt J.F. Biochemistry. 2004; 43: 7307-7327Crossref PubMed Scopus (42) Google Scholar, 24Shilton B. Svergun D.I. Volkov V.V. Koch M.H. Cusack S. Economou A. FEBS Lett. 1998; 436: 277-282Crossref PubMed Scopus (43) Google Scholar). Moreover, ADP release from SecA is stimulated by SecYEG/membrane, raising the question whether SecA retraction from the membrane occurs in the ADP-bound form, the apo form, or both (16Natale P. Swaving J. van der Does C. de Keyzer J. Driessen A.J. J. Biol. Chem. 2004; 279: 13769-13777Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). One approach to resolving questions regarding the timing and stoichiometry of ATP binding and hydrolysis events as well as membrane insertion and retraction events, and how they relate to each other during protein translocation, is to directly measure the kinetic parameters that define the progress of the two reactions and the linkage between them (25Gilbert S.P. Mackey A.T. Methods. 2000; 22: 337-354Crossref PubMed Scopus (67) Google Scholar). Here we report the kinetics of ATP binding, hydrolysis, and product release catalyzed by SecA, measured by pre-steady-state experiments. The results reveal that the SecA dimer rapidly binds and hydrolyzes two ATP molecules in one catalytic turnover (very likely at the high-affinity site), and that the slow step(s) limiting the steady-state turnover rate occur after phosphate release, either before or at ADP release from SecA. Another objective of this study was to identify the catalytic base in the ATPase site that activates a water molecule for attack on the γ phosphate during ATP hydrolysis. Mutation of this residue might allow SecA to bind ATP with high affinity, but not catalyze hydrolysis. Such a mutant could be of value for characterizing the ATP-bound conformation of SecA, which is proposed to insert into the membrane. In a previous report, aspartate 133 (Asp-133) was proposed as the catalytic base, based on the finding that mutation of Asp-133 to asparagine drastically reduced SecA ATPase and protein translocation activity (26Sato K. Mori H. Yoshida M. Mizushima S. J. Biol. Chem. 1996; 271: 17439-17444Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, in the crystal structure of Bacillus subtilis SecA, a homolog of Escherichia coli SecA, this residue does not appear in the correct position to serve a catalytic function (Fig. 1; Glu-131 in B. subtilis SecA); in fact, the structure indicates that glutamate 210 (Glu-210) is a strong candidate for this function (Glu-208 in B. subtilis SecA) (12Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar). We tested the ATPase activities of several glutamate 210 mutants under pre-steady-state conditions and found that SecA-E210N and SecA-E210D are capable of binding ATP with similar affinity as wild type SecA, but their ability to catalyze ATP hydrolysis is severely disrupted, consistent with the hypothesis that Glu-210 is the catalytic base. Proteins and Other Reagents—Wild type SecA (pT7SecA2) was overproduced and purified from E. coli strain BL21.19 as described earlier (6Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar, 27Weinkauf S. Hunt J.F. Scheuring J. Henry L. Fak J. Oliver D.B. Deisenhofer J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 559-565Crossref PubMed Scopus (17) Google Scholar), with the following modifications: an SP-Sepharose column was used instead of Mono-S (protein eluted with 300 mm NaCl in 50 mm Tris-HCl, pH 7.5, 2 mm dithiothreitol) followed by a hexyl-agarose column (protein eluted with 400 mm (NH4)2SO4 in 50 mm Tris-HCl, pH 7.5, 2 mm dithiothreitol). Carboxyl terminus 6 histidine-tagged SecA (His-SecA) was prepared by inserting secA into pET29b in the polycloning site between NdeI/XhoI (pET29b-T7His-SecA), followed by overexpression in BL21.19 cells (growth to 0.6 A600 at 37 °C and a 3-h induction with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside), and purification on a His-Bind resin column (Novagen) according to the manufacturer's protocol (protein eluted with 1 m imidazole in 20 mm Tris-HCl, pH 7.9, 500 mm NaCl). His-tagged SecA-E210D, SecA-E210N, SecA-E210Q, and SecA-E210A were prepared by introducing mutations into pET29b-T7His-SecA using the QuikChange mutagenesis protocol (Stratagene). The mutant proteins were purified by nickel affinity chromatography similar to His-SecA. All SecA proteins were stored in 25 mm Tris-HCl, pH 7.5, 35 mm KCl, 0.5 mm EDTA, 10% glycerol at –80 °C. Protein concentrations were determined by the Bradford assay, absorbance at 280 nm in 6 m guanidinium-HCl, 20 mm potassium phosphate, pH 6.5 (SecA monomer molar extinction coefficient = 71,950 m–1 cm–1), and by amino acid analysis; all three methods yielded values within 20% of each other. Pro-OmpA (28Crooke E. Guthrie B. Lecker S. Lill R. Wickner W. Cell. 1988; 54: 1003-1011Abstract Full Text PDF PubMed Scopus (131) Google Scholar) and urea-treated inverted membrane vesicles (29Rhoads D.B. Tai P.C. Davis B.D. J. Bacteriol. 1984; 159: 63-70Crossref PubMed Google Scholar) were prepared as described. Phosphate-binding protein (PBP) was purified and labeled with 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) as described (30Brune M. Hunter J.L. Corrie J.E. Webb M.R. Biochemistry. 1994; 33: 8262-8271Crossref PubMed Scopus (437) Google Scholar, 31Jeong Y.J. Kim D.E. Patel S.S. J. Biol. Chem. 2002; 277: 43778-43784Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Radioactive nucleotides, [α-32P]ATP and [35S]ATPγS, were purchased from PerkinElmer Life Sciences, [3H]ADP was purchased Amersham Biosciences, [α-32P]ADP was prepared as described previously (32Antony E. Hingorani M.M. Biochemistry. 2003; 42: 7682-7693Crossref PubMed Scopus (81) Google Scholar), and non-radioactive nucleotides were purchased from Sigma. Polyethyleneimine cellulose-F TLC plates were purchased from EM Science and nitrocellulose membranes from Schleicher and Schuell. In Vitro Protein Translocation Assays—For analysis of pro-OmpA translocation, 100-μl reactions were prepared with SecA (50 μg/ml), SecB (66 μg/ml), bovine serum albumin (200 μg/ml), ATP (2 mm), NADH (5 mm), and CK1801.4 urea-treated inverted membrane vesicles (100 μg/ml) in TL buffer (50 mm Tris-HCl, pH 8.0, 50 mm KCl, 5 mm MgCl2). Reactions were initiated with 125I-pro-OmpA (5 × 105 cpm/ml) and pro-OmpA (32 μg/ml) in 50 mm Tris-HCl, pH 8.0, 6 m urea, and incubated for 20 min at 37 °C. Samples were transferred to ice and digested with Proteinase K (1 mg/ml) in the presence or absence of Triton X-100 (0.1%) for 15 min. Proteins were precipitated by addition of 75 μl of 30% trichloroacetic acid, washed with acetone, re-suspended in 50 μl of SDS-PAGE buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 100 mm dithiothreitol, 10% glycerol, 0.1% bromphenol blue), heated for 5 min at 100 °C, followed by electrophoresis on 12% gels and autoradiography. In Vivo Complementation Assays—Complementation was assessed by streaking Bl21.19 cells (containing a temperature-sensitive amber suppressor, an amber mutation in chromosomal secA and a plasmidborne copy of wild type or mutant secA) on media containing 100 μg/ml ampicillin, followed by incubation at permissive (30 °C) or non-permissive temperatures (42 °C) for 16–24 h, and comparison of growth. Nucleotide Binding Assays—ATP binding to SecA proteins was measured by nitrocellulose membrane filtration assays. The membranes were treated with 0.5 n NaOH for 2 min, washed with H2O, and equilibrated in nucleotide binding buffer (50 mm Hepes-NaOH, pH 7.5, 30 mm KCl, 10 mm MgOAc2). 15-μl reactions containing 2.5 μm SecA dimer and 0–250 μm ATP (+0.5 μCi of [α-32P]ATP per point) were incubated in binding buffer for 10 min at 4 °C. 10-μl aliquots of the reaction were filtered through the membrane. The membranes were washed before and after filtration with 150 μl of the binding buffer. One-μl aliquots were spotted onto a separate membrane to measure total nucleotide in the reaction. The molar amount of nucleotide bound to SecA dimer was determined by quantitation on a PhosphorImager (Amersham Biosciences) and plotted versus nucleotide concentration. The binding isotherms were fit to an equation describing 1:1 protein-ligand interaction, [N ·M] = 0.5{(Kd + [Nt] + [Mt]) – [(Kd + [Nt] + [Mt])2 – 4[Nt][Mt]]1/2}, where N ·M is the molar amount of ATP bound to SecA dimer, Nt and Mt are total ATP and SecA concentrations, respectively, and Kd is the apparent dissociation constant. The rate of dissociation of ADP from SecA was measured by incubating 2.5 μm SecA dimer with 250 μm ADP (+0.5 μCi of [α-32P]ADP per point) in nucleotide binding buffer, at 25 °C for 10 min, followed by addition of 10 mm Mg2+-ADP chase and filtration through nitrocellulose membranes at times ranging from 20 s to 10 min. The molar amount of ADP bound to SecA was plotted versus time and fit to a single exponential function to determine the rate constant, koff. Steady-state ATPase Assays—For colorimetric ATPase assays, 25-μl reactions were prepared with SecA (0.2 μm dimer) in buffer (50 mm Hepes-KOH, pH 7.5, 30 mm KCl, 30 mm NH4Cl, 0.5 mm MgOAc2, 1 mm dithiothreitol), in the absence of other ligands (endogenous ATPase) or in the presence of 50 μg of membrane protein/ml of CK1801 inverted membrane vesicle alone (membrane ATPase) or with 20 μm pre-protein PSN (chimera of alkaline phosphatase signal sequence and mature portion of staphylococcal nuclease; translocation ATPase). The reactions were initiated with 4 mm ATP and incubated for 15 min at 37 °C. ATP hydrolysis was measured using the malachite green assay (33Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1821) Google Scholar) with the modifications described previously (6Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar). The steady-state ATPase rate constant was measured by radiometric assays with 1 μm SecA dimer and 1 mm ATP (+ 0.5 μCi of [α-32P]ATP per point) in ATPase buffer (50 mm Hepes, pH 7.5, 30 mm KCl, 10 mm MgOAc2) at 37 °C. At varying times, 5 μl of the reaction was quenched with 5 μl of 0.5 m EDTA and 1-μl aliquots were analyzed by polyethyleneimine cellulose thin-layer chromatography with 0.6 m potassium phosphate, pH 3.4. The molar amount of [α-32P]ADP formed was plotted versus time, and the kcat value was obtained from velocity ÷ [SecA dimer]. Rapid-quench and Pulse-Chase ATPase Assays—Pre-steady-state ATPase assays were performed on a KinTek Corp. quench-flow instrument (Austin, TX) at 37 °C with 6 μm SecA dimer and 1 mm ATP (+ 2 μCi of [α-32P]ATP per point) in ATPase buffer. Sixteen μl of SecA was mixed rapidly with 16 μl of ATP and quenched after varying times (0.004–5 s) with 35 μl of 0.7 m formic acid (final concentrations were 3 μm SecA dimer and 0.5 mm ATP). One-μl aliquots of the reactions were spotted immediately on TLC plates and analyzed as above. Molar amounts of ADP formed were plotted versus time and fit to a single-exponential plus linear equation (burst equation), [ADP] = A(1-e–kt) + Vt, where A and k are burst amplitude and rate constant, respectively, V is steady-state velocity, and t is reaction time. Pulse-chase experiments were performed similarly, except 35 μl of 10–20 mm unlabeled Mg2+-ATP chase was added to the reaction after various times (0.004–2 s). After chase time equivalents to 5–6 turnovers (25 s), the reactions were quenched with 70 μlof0.7 m formic acid and analyzed as above. Stopped-flow experiments were performed on a KinTek Corp. SF-2001 Stopped-Flow instrument (Austin, TX) to measure the rates of ATP hydrolysis and phosphate release catalyzed by SecA. Phosphate (Pi) release was assayed in real-time using fluorescent MDCC-labeled E. coli PBP as described previously (31Jeong Y.J. Kim D.E. Patel S.S. J. Biol. Chem. 2002; 277: 43778-43784Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 34Baird C.L. Gordon M.S. Andrenyak D.M. Marecek J.F. Lindsley J.E. J. Biol. Chem. 2001; 276: 27893-27898Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 35Bertram J.G. Bloom L.B. Hingorani M.M. Beechem J.M. O'Donnell M. Goodman M.F. J. Biol. Chem. 2000; 275: 28413-28420Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Changes in MDCC-PBP fluorescence upon Pi binding were monitored by excitation at 425 nm and monitoring emission above 450 nm (cut-off filter; Corion LL-450 F). A coupled enzyme reaction (Mop) containing 200 μm 7-methylguanosine and 0.03 units/ml purine nucleoside phosphorylase was used in all reactions to sequester contaminant Pi as ribose 1-phosphate, because MDCC-PBP is sensitive to micromolar concentrations of Pi. A Pi calibration curve relating the PBP-MDCC fluorescence signal to Pi concentration was generated prior to each experiment (31Jeong Y.J. Kim D.E. Patel S.S. J. Biol. Chem. 2002; 277: 43778-43784Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). All reactions and syringes were “mopped” for at least 45 min before each experiment. Pi release experiments were carried out at 37 °C in ATPase buffer containing the Mop. A 60-μl solution of 2 μm SecA dimer, incubated with 16 μm MDCC-PBP, was mixed rapidly with an equal volume of 1 mm ATP, and the change in fluorescence monitored over time in the observation cell (final concentrations are 1 μm SecA, 500 μm ATP, 8 μm MDCC-PBP). Background fluorescence, measured for each reaction by omitting Mg2+ to prevent ATP hydrolysis, was subtracted from the traces. Raw data obtained by averaging at least 5 traces were divided by the slope from the Pi calibration curve to determine the molar amount of Pi released during the reaction. Data were fit to a burst equation or a linear equation. The kinetics of ATP binding, hydrolysis, and product release catalyzed by wild type and mutant SecA proteins were measured to better define the mechanism of the ATPase reaction, which is necessary to understand how SecA uses ATP to drive transport of proteins across membranes. The proposed catalytic base, glutamate 210, in the Walker B “DEVD” motif of the high-affinity nucleotide-binding site was replaced by aspartate, asparagine, glutamine, or alanine residues, using site-directed mutagenesis (Fig. 1). A 6-histidine tag was introduced into the mutants to help completely purify these proteins from any contaminating ATPase activity. As a control, a 6-histidine-tagged version of wild type SecA was also prepared and its activity compared with that of untagged SecA; the two wild type SecA proteins functioned similarly in all assays. In a commonly used colorimetric assay used to measure ATP hydrolysis, none of the mutants had any detectable level of activity above background levels (Fig. 2A), alone (endogenous ATPase), or in the presence of inverted membrane vesicles that contain the SecYEG translocon and are stripped of endogenous SecA without (membrane ATPase) or with pre-protein (translocation ATPase) at 37 °C. As expected from previous studies, wild type SecA ATPase activity is stimulated about 10-fold in the presence of SecYEG/membrane and pre-protein (Fig. 2A) (22Zito C.R. Oliver D. J. Biol. Chem. 2003; 278: 40640-40646Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). A more sensitive and accurate radiometric assay used to measure the steady-state rate constants for wild type SecA (kcat = 0.2–0.3 s–1) and mutant SecA endogenous ATPase activity indicates that SecA-E210D retains some level of activity (kcat = 0.15 s–1), but SecA-E210A mutant activity is near background level (kcat = 0.04 s–1) (Fig. 2B). All the mutants, even SecA-E210D, fail to complement inactivated SecA in vivo (Fig. 2B), consistent with the results of an in vitro protein translocation assay in which the mutants cannot catalyze transport of pro-OmpA polypeptide across a membrane (Fig. 2C, lanes 3-6). These initial results suggest that glutamate 210 plays a key role in the ATPase reaction, and therefore the protein translocation activity of SecA. Before this residue can be implicated as the catalytic base, however, some significant caveats must be addressed. Because the steady-state assays described above do not measure individual ATP binding, hydrolysis, and product release steps in the reaction, they do not reveal which of these steps is affected by the mutations, and therefore cannot determine the role of Glu-210 in the catalytic mechanism. In addition, it is possible that the Glu-210 mutations affect SecA function for reasons other than direct disruption of catalytic chemistry (e.g. via some unrelated perturbation of SecA structure). The experiments described below address these concerns. Wild Type SecA as Well as E210D and E210N Mutants Bind Two ATP Molecules Per Dimer—ATP binding to wild type and mutant SecA proteins was examined by nitrocellulose membrane filtration assays. 2.5 μm SecA (dimer concentration) bound 4.8 μm [α-32P]ATP at 4 °C, i.e. a ratio of nearly 2 ATP molecules per SecA dimer (there was no nonspecific ATP binding to the membrane up to 300 μm ATP) (Fig. 3A). The ATP molecules bind SecA with an apparent Kd of 1.4 μm, indicating that the interaction occurs at the high-affinity nucleotide-binding site. No additional ATP binding (i.e. to a low-affinity site on SecA) could be detected at 300 μm ATP, and data at higher ATP concentrations were not considered reliable because of increasing levels of nonspecific ATP binding to the membrane. The SecA-E210D mutant exhibits almost identical nucleotide binding characteristics, with 2 ATP molecules bound per dimer with an apparent Kd of 2.4 μm (Fig. 3B), and SecA-E210N also binds close to 2 ATP per dimer with an apparent Kd of 0.8 μm (Fig. 3C). These data suggest that substitution of glutamate 210 in SecA with aspartate or asparagine does not appreciably affect its nucleotide binding stoichiometry and affinity. In contrast, only a fraction of the SecA-E210A and SecA-E210Q mutant proteins appears capable of binding nucleotide, as their binding isotherms saturate at only ∼1 ATP bound per protein dimer (Fig. 1, Supplemental Materials). Interestingly, a partial proteolysis assay of the proteins reveals that SecA-E210A and SecA-E210Q are also significantly more sensitive to trypsin digestion than wild type SecA and SecA-E210D and SecA-E210N (Fig. 1, Supplemental Materials). These data indicate variations in protein structure/stability between the mutants, and suggest that in the case of the SecA-E210A and SecA-E210Q mutants, the loss of ATPase and protein translocation activity may be attributable to reasons other than disruption of catalytic chemistry. Wild Type SecA Rapidly Binds and Hydrolyzes Two ATP Molecules per Dimer, Unlike the E210D and E210N Mutants—To determine whether mutation of Glu-210 to aspartate or asparagine specifically impacts ATP hydrolysis rather than some other event(s) in the SecA-catalyzed ATPase reaction, it was necessary to directly measure the ATP binding, hydrolysis, and product release steps in the reaction. We first performed rapid quench experiments with 2.6–3 μm wild type SecA dimer and 500 μm [α-32P]ATP, and observed a burst of ATP hydrolysis in the first turnover at a rate constant of 27 ± 2.5 s–1, followed by a slow linear phase at a rate constant of 0.3 s–1 (similar to the steady-state kcat of 0.25 s–1) (Fig. 4A). The amplitude of the burst phase is 5.2 ± 0.1 μm, close to 2 ATP molecules per SecA dimer. Next, a pulse-chase experiment was performed to measure the kinetics of ATP binding to SecA. The protein (2.6–3 μm dimer) was mixed with [α-32P]ATP (500 μm) for varying times (mixing time), and chased with 20-fold excess unlabeled Mg2+-ATP for time equivalent to 5–6 turnovers (chase time). During the chase, bound [α-32P]ATP may be hydrolyzed to [α-32P]ADP + Pi or remain unhydrolyzed (either bound to SecA or released into solution). Any free [α-32P]ATP in solution is diluted upon addition of unlabeled ATP chase and is not available for further binding and hydrolysis. Thus, the pulse-chase experiment measures the rate of ATP binding to SecA and the fraction of the SecA·ATP complex that undergoes hydrolysis. The data reveal a burst of A" @default.
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• W2008875470 date "2005-04-01" @default.
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• W2008875470 title "Role of a Conserved Glutamate Residue in the Escherichia coli SecA ATPase Mechanism" @default.
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• W2008875470 doi "https://doi.org/10.1074/jbc.m414224200" @default.
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