Sequential Hydrolysis of ATP Molecules Bound in Interacting Catalytic Sites of Escherichia coli Transcription Termination Protein Rho
1998; Elsevier BV; Volume: 273; Issue: 41 Linguagem: Inglês
10.1074/jbc.273.41.26477
ISSN1083-351X
Autores Tópico(s)DNA Repair Mechanisms
ResumoEscherichia coli transcription termination protein Rho, an RNA-dependent ATPase, disrupts transcription complexes, releasing RNA and allowing RNA polymerase to recycle. Homohexameric Rho binds three molecules of MgATP in a single class of catalytically competent sites. In rapid mix chemical quench experiments, when Rho saturated with ATP was mixed with RNA and the reaction was quenched after various times, hydrolysis of the three bound ATP molecules was not simultaneous. A hydrolysis burst of one molecule of ATP per hexamer occurred at >300 s−1, followed by steady-state hydrolysis at 30 s−1per hexamer. The burst also shows that a step following ATP hydrolysis is rate-limiting for overall catalysis and requires communication among the three catalytic sites during net ATP hydrolysis. The rate of hydrolysis of radiolabeled ATP when one labeled and two unlabeled ATP molecules are bound indicates a sequential pattern of hydrolysis. Positive cooperativity of catalysis occurs among the catalytic sites of Rho; when only one ATP molecule is bound per hexamer, ATP hydrolysis upon addition of RNA is 30-fold slower than when ATP is saturating. These behaviors are comparable to those of F1-type ATPases, with which Rho shares a number of structural features. Escherichia coli transcription termination protein Rho, an RNA-dependent ATPase, disrupts transcription complexes, releasing RNA and allowing RNA polymerase to recycle. Homohexameric Rho binds three molecules of MgATP in a single class of catalytically competent sites. In rapid mix chemical quench experiments, when Rho saturated with ATP was mixed with RNA and the reaction was quenched after various times, hydrolysis of the three bound ATP molecules was not simultaneous. A hydrolysis burst of one molecule of ATP per hexamer occurred at >300 s−1, followed by steady-state hydrolysis at 30 s−1per hexamer. The burst also shows that a step following ATP hydrolysis is rate-limiting for overall catalysis and requires communication among the three catalytic sites during net ATP hydrolysis. The rate of hydrolysis of radiolabeled ATP when one labeled and two unlabeled ATP molecules are bound indicates a sequential pattern of hydrolysis. Positive cooperativity of catalysis occurs among the catalytic sites of Rho; when only one ATP molecule is bound per hexamer, ATP hydrolysis upon addition of RNA is 30-fold slower than when ATP is saturating. These behaviors are comparable to those of F1-type ATPases, with which Rho shares a number of structural features. adenosine 5′-O-(γ-thio)triphosphate TNP-ADP, the 2′,3′-O-(2,4,6-trinitrophenyl) derivatives of ATP and ADP. Escherichia coli transcription termination protein Rho aids in the release of newly synthesized RNA from paused transcription complexes (reviewed in Ref. 1Richardson J.P. Greenblatt J. Neidhardt F.C. Escherichia coli and Salmonella. American Society for Microbiology, Washington, D. C.1996: 822-848Google Scholar). The homohexameric protein binds nascent RNA and, with the RNA-dependent hydrolysis of ATP, disrupts the ternary transcription complex, releasing product RNA and allowing RNA polymerase to recycle. The discovery of a 5′ → 3′ RNA-DNA helicase activity of Rho (2Brennan C.A. Dombroski A.J. Platt T. Cell. 1987; 48: 945-952Abstract Full Text PDF PubMed Scopus (186) Google Scholar) suggested that Rho might disrupt the RNA-DNA duplex of the transcription bubble. Recent studies of ternary transcription complexes (Refs. 3Nudler E. Mustaev A. Lukhtanov E. Goldfarb A. Cell. 1997; 89: 33-41Abstract Full Text Full Text PDF PubMed Google Scholar, 4Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Crossref PubMed Scopus (300) Google Scholar, 5Komissarova N. Kashlev M. J. Biol. Chem. 1997; 272: 15329-15338Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar and reviewed in Ref. 6Landick R. Cell. 1997; 88: 741-744Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) suggest that such disruption could be important in transcription termination, as could be the release of the nascent RNA just 5′ of the RNA-DNA duplex from its interactions with RNA polymerase. As described by Nudler et al. (7Nudler E. Avetissova E. Markovtsov V. Goldfarb A. Science. 1996; 273: 211-217Crossref PubMed Scopus (176) Google Scholar), the interaction of RNA with RNA polymerase immediately 5′ from the RNA-DNA hybrid may control the opening and closing of an RNA polymerase clamp around the DNA template near the leading edge of the enzyme, and contribute to the stability of the ternary transcription complex. An appealing model for Rho is one in which the enzyme binds to exposed mRNA behind RNA polymerase and travels 5′ → 3′ along the RNA as it hydrolyzes ATP, binding and releasing RNA from different parts of the hexamer to accomplish movement (8Walstrom K.M. Dozono J.M. von Hippel P.H. Biochemistry. 1997; 36: 7993-8004Crossref PubMed Scopus (47) Google Scholar). Such activity could release nascent RNA from RNA polymerase-binding sites and could constitute the basis for its RNA-DNA helicase activity, both of which might be involved in transcript release from paused ternary transcription complexes. The finding that the same number of ATP molecules per RNA length is hydrolyzed by Rho traveling along RNA and Rho unwinding RNA-DNA hybrids (8Walstrom K.M. Dozono J.M. von Hippel P.H. Biochemistry. 1997; 36: 7993-8004Crossref PubMed Scopus (47) Google Scholar) supports this hypothesis. Rho binds single-stranded RNA, showing preferred entry regions on RNA upstream of eventual transcription termination sites. However, the characteristics of these regions, beyond low secondary structure and some preference for a C-rich, G-poor base composition (9Alifano P. Rivellini F. Limauro D. Bruni C.B. Carlomagno M.S. Cell. 1991; 64: 553-563Abstract Full Text PDF PubMed Scopus (109) Google Scholar), are too poorly understood to permit their identification by sequence inspection. When bound to RNA, Rho protects 80 bases from ribonuclease degradation (10Galluppi G.R. Richardson J.P. J. Mol. Biol. 1980; 138: 513-539Crossref PubMed Scopus (102) Google Scholar, 11Bear D.G. Hicks P.S. Escudero K.W. Andrews C.L. McSwiggen J.A. von Hippel P.H. J. Mol. Biol. 1988; 199: 623-635Crossref PubMed Scopus (83) Google Scholar). The binding of Rho to 10-base RNA oligomers was reported as best fit by three tight and three weaker sites per hexamer (12Geiselmann J. Yager T.D. von Hippel P.H. Protein Sci. 1992; 1: 861-873Crossref PubMed Scopus (41) Google Scholar, 13Wang Y. von Hippel P.H. J. Biol. Chem. 1993; 268: 13947-13955Abstract Full Text PDF PubMed Google Scholar). The RNA-dependent hydrolysis of ATP is essential for the transcription termination function of Rho. Two components of ternary transcription complexes, the DNA template and RNA polymerase, are not required to elicit this ATPase activity, thus considerably simplifying study of the reaction (14Lowery-Goldhammer C. Richardson J.P. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2003-2007Crossref PubMed Scopus (93) Google Scholar). The reaction is particularly well stimulated by the RNA homopolymer poly(C), and Rho is frequently assayed in vitro by measuring its poly(C)-dependent ATPase activity. Previous work has shown that the Rho hexamer binds three molecules of MgATP in a single class of catalytically competent sites (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar, 16Geiselmann J. von Hippel P.H. Protein Sci. 1992; 1: 850-860Crossref PubMed Scopus (59) Google Scholar). An additional class of three ATP-binding sites of lower affinity has also been suggested (16Geiselmann J. von Hippel P.H. Protein Sci. 1992; 1: 850-860Crossref PubMed Scopus (59) Google Scholar), although the catalytic activity of these sites was not assessed. The stoichiometry for ATP and RNA oligomer interactions with Rho, together with studies of Rho quaternary structure (17Geiselmann J. Yager T.D. Gill S.C. Calmettes P. von Hippel P.H. Biochemistry. 1992; 31: 111-121Crossref PubMed Scopus (77) Google Scholar, 18Geiselmann J. Seifried S.E. Yager T.D. Liang C. von Hippel P.H. Biochemistry. 1992; 31: 121-132Crossref PubMed Scopus (55) Google Scholar, 19Horiguchi T. Miwa Y. Shigesada K. J. Mol. Biol. 1997; 269: 514-528Crossref PubMed Scopus (31) Google Scholar), have led to a proposed model of Rho as a trimer of dimers in which adjacent identical subunits may alternate in their ability to bind and hydrolyze ATP and to bind and release RNA (8Walstrom K.M. Dozono J.M. von Hippel P.H. Biochemistry. 1997; 36: 7993-8004Crossref PubMed Scopus (47) Google Scholar, 15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar, 20Geiselmann J. Wang Y. Seifried S.E. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7754-7758Crossref PubMed Scopus (101) Google Scholar). Our goal is to elucidate the molecular mechanism of Rho activity, including the sequence of ATP and RNA binding, ATP hydrolysis, product release, and interactions with other molecules that sum to transcription termination. Travel in a 5′ → 3′ direction along single-stranded RNA can readily be seen as consistent with the hydrolysis in an ordered sequence of ATP molecules that are bound to Rho, but evidence for such a hydrolysis pattern is lacking. We present the results of rapid mix chemical quench experiments and isotope partitioning studies, which show that hydrolysis of the three bound ATP molecules is sequential. These results also indicate communication among the active sites of Rho. In addition, we show catalytic cooperativity among Rho active sites. Wild type Rho fromE. coli was purified as described (21Mott J.E. Grant R.A. Ho Y.-S. Platt T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 88-92Crossref PubMed Scopus (142) Google Scholar) from strain AR120/A6 containing plasmid p39ASE (22Nehrke K.W. Seifried S.E. Platt T. Nucleic Acids Res. 1992; 20: 6107Crossref PubMed Scopus (25) Google Scholar). The concentration of Rho was spectrophotometrically determined using ε280 nm1% = 3.25 cm−1 (17Geiselmann J. Yager T.D. Gill S.C. Calmettes P. von Hippel P.H. Biochemistry. 1992; 31: 111-121Crossref PubMed Scopus (77) Google Scholar). The enzyme preparation used for most experiments had a specific activity with poly(C) at 37 °C of 12–15 units mg−1. Some experiments were repeated with an independent enzyme preparation that had a specific activity of 22–26 units mg−1. These values are in the range of specific activities reported in the literature (10–30 units mg−1). A preparation of Rho E155K with a specific activity at 37 °C of 18 units mg−1 was also used. All Rho preparations appeared >95% pure on Coomassie Blue-stained SDS-polyacrylamide gels. A unit of activity is that amount of enzyme that hydrolyzes 1 μmol of ATP in 1 min. ATPγS1 was from Boehringer Mannheim, and [35S]ATPγS, 1250 Ci/mmol, was from NEN Life Science Products (NEG027H). Radioactive ATPγS was diluted to 42,000 cpm nmol−1 before use. TNP-ATP was synthesized and purified according to Hiratsuka and Uchida (23Hiratsuka T. Uchida K. Biochim. Biophys. Acta. 1973; 320: 635-647Crossref PubMed Scopus (143) Google Scholar). [γ-32P]TNP-ATP was synthesized and purified as described by Grubmeyer and Penefsky (24Grubmeyer C. Penefsky H.S. J. Biol. Chem. 1981; 256: 3718-3727Abstract Full Text PDF PubMed Google Scholar). The concentrations of solutions of TNP-ATP were determined in Tris-HCl or Tris acetate buffer at pH 8 using ε408 nm = 2.64 × 104m−1 cm−1 (23Hiratsuka T. Uchida K. Biochim. Biophys. Acta. 1973; 320: 635-647Crossref PubMed Scopus (143) Google Scholar). [γ-32P]ATP at a specific activity of 1–10 Ci/mmol was purchased from NEN Life Science Products or synthesized from [32Pi] and ATP according to the exchange method of Glynn and Chappell (25Glynn I.M. Chappell J.B. Biochem. J. 1964; 90: 147-149Crossref PubMed Scopus (1258) Google Scholar) as modified by Grubmeyer and Penefsky (24Grubmeyer C. Penefsky H.S. J. Biol. Chem. 1981; 256: 3718-3727Abstract Full Text PDF PubMed Google Scholar). Poly(C) was from Miles Laboratories; poly(U) was from Boehringer Mannheim, ICN, or Amersham Pharmacia Biotech; and poly(A) was from Boehringer Mannheim. Each was dissolved in water to 2 or 10 mg/ml. [18O]H2O from Isotech, Inc., was 98.5 atom %. D2O was from Aldrich. TAGME buffer is 40 mm Tris acetate, pH 8.3, at room temperature (21–24 °C), 150 mm potassium glutamate, 1 mm magnesium acetate, 0.1 mm EDTA. V max, K m, ATP binding, and isotope partitioning measurements were carried out as in Stitt (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar) except that for most experiments the buffer was 40 mm Tris acetate and 150 mm potassium glutamate rather than 40 mm Tris-HCl and 50 mm KCl, and many experiments were conducted at room temperature to facilitate comparison with rapid mix results; exceptions are noted. To separate free from bound ATP in binding experiments, a Microcon-10 ultrafiltration apparatus was used (Amicon). In control samples from which Rho was omitted, 3–5% of the adenine nucleotide bound to the apparatus; all measurements of bound and free nucleotide were corrected for this adventitious binding. For V maxmeasurements at room temperature, assays in general were carried out in 100 μl volumes that were 0.5–1 mm in adenine nucleoside triphosphate and contained 1–2 μg/ml poly(C), and the amount of Pi product was determined following an acid quench and charcoal absorption of adenine nucleotides from the entire reaction volume, as described below. To measure rates for the slowest reactions, 200–300-μl mixtures were prepared and multiple 10–20-μl samples removed for Pi determinations at various times; rates were obtained from linear portions of plots of nucleotide hydrolyzedversus time. Experiments with ATPγS were conducted with 5-fold more Rho than usual (2.5 rather than 0.5 μg/ml in the assay) and for 40 min (rather than the standard 20 min at room temperature); assays involving poly(U) and ATP used 10-fold higher than normal concentrations of both Rho and poly(U) and were for 40 min; for TNP-ATP with poly(U) and ATP with poly(A), final concentrations of 1.33 mg/ml Rho and 300 μg/ml polynucleotide were used, with multiple Pi determinations between 0 and 30 min after addition of Rho; and for poly(A) with TNP-ATP, 1.33 mg/ml enzyme and 1.5 mg/ml polynucleotide were used, with samples taken at hourly intervals to 6 h. In all cases, similar reaction mixtures lacking either enzyme or RNA served as controls. The rate of ATP hydrolysis by Rho in the absence of RNA was measured using Rho at 1.76 μg/ml in TAGME buffer with 200 μm[γ-32P]ATP at 20,000 cpm/nmol. At t = 0 and at 30–60-min intervals to t = 2 h, the amount of 32Pi was determined in 50-μl samples following charcoal absorption of adenine nucleotides (described below). To assay for a burst of ATP hydrolysis in the absence of RNA, 90-μl mixtures were prepared, each containing 10 μm[γ-32P]ATP at 660,000 cpm/nmol (with 3.3% background32Pi radioactivity) or 0.5 mm[γ-32P]TNP-ATP at 1500 cpm/nmol (0.5% background Pi counts). The final component added to control tubes was 10 μl of buffer; to other tubes, 133 μg of Rho (468 pmol hexamer) was added in a volume of 10 μl. 250 μl of 5% w/v trichloroacetic acid quench was mixed with the contents of each tube 2–3 s after the addition of the final component, followed by 150 μl of acid-washed charcoal (80 mg/ml in water). For ATP, a second sample was quenched 60 s after addition of the final component. After pelleting the charcoal with bound adenine nucleotides by centrifugation in a microcentrifuge at 12,000 × g for 2 min, 200 μl of the supernatant was removed and the amount of32Pi determined by liquid scintillation. These experiments were carried out using an Update Instruments model 1010 with a grid-type mixer of 1.6-μl volume. Experiments were at room temperature (21–23 °C) or at 4 °C and were conducted in TAGME buffer. Two experimental designs were used. In the first, Rho + [γ-32P]ATP were present in one syringe, and RNA in the second; alternatively, Rho alone was in one syringe, with RNA + radiolabeled nucleoside triphosphate in the second. In the first experimental design, the results were corrected for the slow Rho-catalyzed hydrolysis of [γ-32P]ATP in the absence of RNA, which was significant at the high concentration of Rho employed. The second experimental configuration gave similar results but did not involve such a correction and was preferentially used. In general, approximately 100 μl of mixed, aged reactants (50 μl from each syringe) were ejected from the aging hose into 400 μl of 5% w/v trichloroacetic acid quench. 50 μl of the quenched mix was taken to determine total radioactivity and thus the volume that was actually quenched. 350 μl of the quenched mix was assayed for [32Pi] (or, in the case of ATPγS, [35S] thiophosphate) following charcoal absorption of adenine nucleotides as described above. The concentrations of reactants in the mixer in most experiments were as follows: Rho, 0.88 mg/ml (10 μm active sites); [γ-32P]ATP or its analogs, 0.1 mm (the equivalent of 30 ATP molecules per Rho hexamer); RNA, 0.2 mg/ml (600 μm bases; thus two 100-base lengths of RNA per hexamer). These values were chosen so that the ATP concentration was sufficiently high that its on-rate (estimated fromk cat /K m)was not limiting, and the enzyme concentration was such that turnover of a single ATP per hexamer would produce product 32Pisignificantly above the background level of32Pi in the nucleoside triphosphate substrates. In one experiment with TNP-ATP and poly(C), tripling the nucleoside triphosphate concentration while doubling those of enzyme and RNA produced similar results. Aging times (the times elapsed between mixing and quenching) ranged from 2.7 ms to 1–2 s for ATP with poly(C), TNP-ATP with poly(C), and in low ATP with chase experiments, and 2.7 ms to 60 s for ATPγS with poly(C), ATP with poly(U), TNP-ATP with poly(U), and at low ATP levels. These experiments, using 0.35 μm Rho hexamer and 0.17–6.0 μm[γ-32P]ATP at 511 cpm/pmol, were carried out as in Stitt (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar), except that in most cases TAGME buffer was used, and a 50-μl sample was injected into 150 μl of chase solution, followed by 50 μl of 50% w/v trichloroacetic acid as quenching agent. ATP hydrolysis was monitored as above by measuring the production of [32Pi] from [γ-32P]ATP following charcoal precipitation of adenine nucleotides. The same binding mixture was used for both binding and isotope partitioning measurements. In experiments with [γ-32P]TNP-ATP, the ligand was 0.19–5.9 μm at 1075 cpm/pmol. 0.5-ml reactions were prepared in a final concentration of 80% [18O]H2O in TAGME buffer; several different protocols were used as follows. 1) For V maxconditions, the reaction contained 1 μg of Rho, 0.4 μg of poly(C), and was 2 mm in ATP, 3 mm in magnesium acetate. This reaction was quenched after 30 min at 37 °C, when about 60% of the ATP had been hydrolyzed, by addition of EDTA to 10 mm. As a control, an identical mixture lacking Rho was also prepared. 2) A reaction at a low ATP concentration of 5 μm was prepared similarly to the V max reaction, with the addition of an ATP-regenerating system consisting of phosphoenolpyruvate at 1 mm plus 5 μg of pyruvate kinase. This reaction was incubated at 37 °C for 3 h prior to addition of a similar EDTA quench. 3) To detect net reversal of ATP hydrolysis, a mixture similar to the first was prepared, with ATP replaced by 2 mm ADP plus 2 mm Pi. This mixture was incubated at 37 °C for 5.5 h prior to addition of the EDTA quench. Following quenching, each reaction mixture was filtered through an Amicon Microcon 10 ultrafiltration apparatus and was stored at −20 °C until analysis. At this time the samples were thawed, supplemented with D2O to 10% v/v, and analyzed by one-dimensional 31P NMR with proton decoupling using a Brüker DRX300 spectrometer operating at 121.5 MHz. The E. coli Rho protein used in an earlier determination of the kinetic mechanism (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar) is now known to carry the mutation E155K (22Nehrke K.W. Seifried S.E. Platt T. Nucleic Acids Res. 1992; 20: 6107Crossref PubMed Scopus (25) Google Scholar, 26Platt T. Richardson J.P. McKnight S.L. Yamamoto K.R. Transcriptional Regulation. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 365-388Google Scholar); this mutation lies in a proposed hinge region of Rho between the RNA-binding amino terminus and the ATP-binding domain (27Opperman T. Richardson J.P. J. Bacteriol. 1994; 176: 5033-5043Crossref PubMed Google Scholar). Parameters important to the present work were therefore remeasured for the true wild type Rho (Rho+) that was used in these studies. (A more complete study of adenine nucleotide interactions with RhoE155K was published earlier (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar).) Table I shows data obtained at 22 °C for the two types of Rho using poly(C) as the RNA cofactor; values fork cat , K m,K D, and n values for binding and isotope partitioning are unaffected by the mutation. Rho+ and RhoE155K also have similar K m values at 37 °C for ATP, 8–10 μm, and similar hexamer V maxvalues with poly(C). A notable feature of both types of Rho is the ∼10-fold tighter binding of ATP in chloride- versusglutamate-containing buffer. This difference is also seen in isotope partitioning experiments: in chloride-containing buffer all bound ATP molecules are hydrolyzed upon addition of RNA, but in glutamate-containing buffer only ∼70% of bound ATP molecules are hydrolyzed because of a faster off-rate. (The off-rates in Table II are calculated, minimum values; we note that to explain the isotope partitioning results,k off in glutamate buffer must be slightly faster than the calculated 6.6 s−1.)Table IProperties of Rho and RhoE155K at 22 °CEnzyme Rho+Enzyme RhoE155KTKMETAGMETKMETAGMEk cat10 s−110 s−110 s−110 s−1K m ATP7 μm3 μmk cat/K m1.4 × 106m−1 s−13.3 × 106m−1 s−1K DATP0.4 μm2 μm0.2 μm4 μmk off0.56 s−16.6 s−1ATP-bound/ hexamer3333IPP32–2.52.82.6Buffer TKME includes 50 mm KCl; buffer TAGME includes 150 mm potassium glutamate. See "Materials and Methods."Per active site; calculated from V maxATPase measurements assuming three independent active sites/hexamer.Calculated from the measured K D values using k cat/K m as a minimal value for k on.IPP, results of isotope partitioning experiments in which Rho was initially saturated with [γ-32P]ATP; moles of product of 32Pi per hexamer. RhoE155K data in TKME are at 37 °C (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab Table IIResults of rapid mix chemical quench experimentsNucleotideRNASubstrate hydrolysis burst; nucleotides/hexamerSteady state rate; nucleotides s−1/hexamernATPPoly(C)0.7–1.3305ATP, 4 °CPoly(C)0.54.51TNP-ATPPoly(C)0.4–0.862ATPPoly(U)0.60.161TNP-ATPPoly(U)0.4–0.50.062ATPγSPoly(C)0–0.10.82Rho plus nucleotide was mixed with RNA or Rho was mixed with RNA plus nucleotide to initiate the reaction, which was then quenched in acid after various times. Nucleotides labeled in the γ-phosphoryl group were used, and their hydrolysis was quantitated by measurement of the amount of labeled phosphate product in quenched samples (see "Materials and Methods").Number of times the experiment was performed. Open table in a new tab Buffer TKME includes 50 mm KCl; buffer TAGME includes 150 mm potassium glutamate. See "Materials and Methods." Per active site; calculated from V maxATPase measurements assuming three independent active sites/hexamer. Calculated from the measured K D values using k cat/K m as a minimal value for k on. IPP, results of isotope partitioning experiments in which Rho was initially saturated with [γ-32P]ATP; moles of product of 32Pi per hexamer. RhoE155K data in TKME are at 37 °C (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar). Rho plus nucleotide was mixed with RNA or Rho was mixed with RNA plus nucleotide to initiate the reaction, which was then quenched in acid after various times. Nucleotides labeled in the γ-phosphoryl group were used, and their hydrolysis was quantitated by measurement of the amount of labeled phosphate product in quenched samples (see "Materials and Methods"). Number of times the experiment was performed. For both types of Rho, our experiments indicated 3 ATP molecules bound per hexamer (Fig. 1) and did not show a second class of binding sites with lower affinity for ATP (16Geiselmann J. von Hippel P.H. Protein Sci. 1992; 1: 850-860Crossref PubMed Scopus (59) Google Scholar). If data from ultrafiltration binding experiments were not corrected for ATP bound to the apparatus in the absence of Rho, then, as shown in theinset to Fig. 1, an additional class of ATP-binding sites appears to exist. These "sites" apparently represent adventitious binding of ATP to the apparatus, rather than a second class of sites on Rho. Earlier work demonstrated three adenine nucleotide-binding sites on Rho both in the absence and presence of RNA (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar). Previously (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar), the rate of ATP hydrolysis by RhoE155K at 37 °C in the absence of RNA was estimated to be no greater than 2 ATP molecules/hexamer/h. Additional measurements showed that the rate at 22 °C for Rho+ is linear, with values from 1.5 to 4.4 × 10−4 s−1 hexamer−1, 100,000-fold slower than the poly(C)-stimulated V max (data not shown). In addition, samples taken within 5 s after the addition of Rho to mixtures containing either [γ-32P]ATP or the ribose-modified ATP analog [γ-32P]TNP-ATP showed no burst of32Pi formation (see "Materials and Methods"). Thus ATP hydrolysis on the enzyme in the absence of RNA is slow, with no evidence for rapid on-enzyme hydrolysis of bound ATP molecules followed by slow product release. To gain further understanding of the molecular mechanism by which Rho uses the energy of ATP hydrolysis, we wanted to determine whether the ATP molecules bound in catalytic sites are hydrolyzed simultaneously or sequentially upon RNA binding. To address this question we performed rapid mix experiments employing chemical quench techniques (28Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (194) Google Scholar). In these experiments, components of a reaction are separately loaded into two syringes that are connected to a small grid-type mixing chamber with an outlet to an aging hose. A ram drives both syringe plungers simultaneously, forcing portions of the contents of the syringes through the 1.6-μl mixer and into the aging hose. After the mixed reactants have aged for a defined time, a volume of the reaction is quenched with acid and then assayed for product to determine the extent of the reaction. A notable feature of this technique is that it measures the sum of products that are enzyme-bound plus those that have been released into solution. When Rho was mixed with excess poly(C) and [γ-32P]ATP using this apparatus (see "Materials and Methods"), 1 mol of ATP per mol of Rho hexamer was hydrolyzed at the fastest quench time, 2.7 ms after mixing (Fig. 2; Table II). This burst was followed by steady-state hydrolysis atV max (which was measured using the same sample in separate experiments). Two independent preparations of Rho and several batches of [γ-32P]ATP gave similar results. Since Rho has three active sites per hexamer, a hydrolysis burst of one ATP per hexamer could result from irreversible hydrolysis of ATP at only one of the three sites. Alternatively, the burst could reflect the value of an on-enzyme equilibrium between substrates and products at each active site (prior to product release) whose final position is equivalent to 0.33 ATP hydrolyzed per site (28Johnson K.A. Methods Enzymol. 1995; 249: 38-61Crossref PubMed Scopus (194) Google Scholar). The results of several experiments excluded this alternative explanation. Establishment of an equilibrium requires the reversible hydrolysis of ATP in the active site. Such an equilibrium is very unlikely given the reported absence of on-enzyme intermediate Pi/H2O oxygen exchange for RhoE155K (15Stitt B.L. J. Biol. Chem. 1988; 263: 11130-11137Abstract Full Text PDF PubMed Google Scholar). However, to test the wild type enzyme, the phosphate product from reactions in which Rho hydrolyzed [γ-16O3]ATP in 18O water was analyzed for 18O content by NMR. At least one solvent18O atom must be incorporated into product Piduring hydrolysis in [18O]H2O. On-enzyme reversibility of the chemistry would be indicated by the production of phosphate with more than one 18O atom. Fig. 3 shows the NMR spectrum of the phosphate product from a V max reaction; no phosphate molecules with more than one 18O atom were found (less than 5% excess incorporation would have been easily detected). The same result was found in an experiment carried out at 5 μm ATP in the presence of an ATP-regenerating system, indicating that no different behavior occurred under non-saturating substrate conditions. We further tested for reversibility of ATP hydrolysis by Rho by incubating the enzyme with RNA, ADP, and Pi in [18O]H2O-containing buffer for more than 5 h, followed by analysis of Pi for 18O content. In this experiment, no 18O-containing phosphate was found. We therefore conclude that ATP hydrolysis by Rho is irreversible. Our rapid mix results demonstr
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