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• W2010742469 abstract "The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E·Ile-AMP; Scheme FS1). Isoleucine (Ile) binds to the E·ATP complex (K 4 = 1.7 ± 0.9 μm) ∼35-fold more tightly than to E(K 1 = 50–100 μm), primarily due to a reduction in the Ile dissociation rate constant (k -1 ≈ 100–150 s−1,cf. k -4 = 3 ± 1.5 s−1). Similarly, ATP binds more tightly toE·Ile (K 3 = ∼70 μm) than to E (K 2 = ∼2.5 mm). The formation of the E·isoleucyl adenylate intermediate, E·Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k +5 = ∼60 s−1) and the reverse rate constant (k -5 = ∼150–200 s−1) to be determined from pyrophosphorolysis of a pre-formed E·Ile-AMP complex (K 6= ∼0.25 mm). Scheme FS1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme FS1could also provide an adequate description of the kinetics of tRNA aminoacylation (k cat,tr = ∼0.35 s−1) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS. The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E·Ile-AMP; Scheme FS1). Isoleucine (Ile) binds to the E·ATP complex (K 4 = 1.7 ± 0.9 μm) ∼35-fold more tightly than to E(K 1 = 50–100 μm), primarily due to a reduction in the Ile dissociation rate constant (k -1 ≈ 100–150 s−1,cf. k -4 = 3 ± 1.5 s−1). Similarly, ATP binds more tightly toE·Ile (K 3 = ∼70 μm) than to E (K 2 = ∼2.5 mm). The formation of the E·isoleucyl adenylate intermediate, E·Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k +5 = ∼60 s−1) and the reverse rate constant (k -5 = ∼150–200 s−1) to be determined from pyrophosphorolysis of a pre-formed E·Ile-AMP complex (K 6= ∼0.25 mm). Scheme FS1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme FS1could also provide an adequate description of the kinetics of tRNA aminoacylation (k cat,tr = ∼0.35 s−1) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS. aminoacyl-tRNA synthetase pseudomonic acid A isoleucyl-tRNA synthetase adenosine 5′-tetraphospho-5′-adenosine. Activation of the carboxyl group of amino acids with ATP to form the corresponding aminoacyl adenylate prior to transfer to tRNA is the first step in protein biosynthesis. The activation of each amino acid is catalyzed by a specific aminoacyl-tRNA synthetase (aaRS)1 which first catalyzes the activation (and stabilization) of the amino acid as a mixed anhydride adenylate, and the subsequent acyl transfer to the corresponding cognate tRNA (for a review, see Ref. 1Meinnel T. Mechulam Y. Blanquet S. Soll D. RajBhandary U.L. tRNA, Structure, Biosynthesis and Function. ASM Press, Washington, D. C.1995: 251-292Google Scholar). The high level of phylogenic divergence between these enzymes in prokaryotes and eukaryotes (2Eriani G. Delarue M. Poch O. Gangloff J. Moras D. Nature. 1990; 347: 203-206Crossref PubMed Scopus (1190) Google Scholar, 3Lamour V. Quevillon S. Diriong S. N′Guyen V.C. Lipinski M. Mirande M. Proc. Natl. Acad. Sci. U. S. A. 1994; 9l: 8670-8674Crossref Scopus (135) Google Scholar, 4Rauhut R. Gabius H-J. Cramer F. Biosystems. 1986; 19: 173-183Crossref PubMed Scopus (5) Google Scholar, 5Nagel G.M. Doolittle R.F. J. Mol. Evol. 1995; 40: 487-498Crossref PubMed Scopus (109) Google Scholar, 6Carter Jr., C.W. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (329) Google Scholar), together with their essential role in protein synthesis, makes aminoacyl-tRNA synthetases excellent targets for the development of selectively acting anti-bacterial agents. The best known example of such a compound is pseudomonic acid A (PS-A) which specifically inhibits bacterial isoleucyl-tRNA synthetase (IleRS) around 10,000-fold more potently that the corresponding mammalian enzyme (7,8) and is a highly effective antibiotic. Staphylococcus aureus infections, particularly of the respiratory tract, are a major clinical problem and, because of the emergence of resistance to “classical” antibiotics (e.g. β-lactams), this organism is an important target for the development of new antibiotics. However, resistance to PS-A itself is emerging although not yet a clinically relevant problem (e.g. Refs. 9Dyke K.G.H. Curnock S.P. Golding M. Noble W.C. FEMS Microbiol. Lett. 1991; 77: 195-198Crossref Scopus (41) Google Scholar and 10Farmer T.H. Gilbart J. Elson S. J. Antimicrobial Chemotherapy. 1992; 30: 587-596Crossref PubMed Scopus (53) Google Scholar). Therefore, we have considerable interest in understanding the reaction cycle and inhibition of S. aureus IleRS by PS-A in order to aid the design of novel inhibitors and to understand the mechanisms of PS-A resistance. Following overexpression of S. aureus IleRS (11Chalker A. Ward J. Fosberry A. Hodgson J. Gene (Amst.). 1994; 141: 103-108Crossref PubMed Scopus (22) Google Scholar), reagent quantities of this enzyme are now available. In this paper, we describe the construction of a minimal reaction mechanism for amino acid activation by IleRS which adequately describes all of the steady-state and transient kinetic properties of this enzyme. Accompanying papers describe a detailed characterization of the kinetics and mechanism of inhibitor binding (12Pope A.J. Moore K.J. McVey M. Mensah L. Benson N. Osbourne N. Broom N. Brown M.J.B. O’Hanlon P. J. Biol. Chem. 1998; 273: 31691-31701Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and an analysis of the effects of ligand binding upon proteolysis of IleRS (13Pope A.J. McVey M. Fantom K. Moore K.J. J. Biol. Chem. 1998; 273: 31702-31706Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The preparation of IleRS (12Pope A.J. Moore K.J. McVey M. Mensah L. Benson N. Osbourne N. Broom N. Brown M.J.B. O’Hanlon P. J. Biol. Chem. 1998; 273: 31691-31701Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and the methods used for steady-state ATP/PPi exchange and tRNA aminoacylation reactions (15Calendar R. Berg P. Biochemistry. 1966; 5: 1681-1690Crossref PubMed Scopus (160) Google Scholar) were as described in the accompanying paper (12Pope A.J. Moore K.J. McVey M. Mensah L. Benson N. Osbourne N. Broom N. Brown M.J.B. O’Hanlon P. J. Biol. Chem. 1998; 273: 31691-31701Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Isoleucinyl adenylate (Ile-ol-AMP; Fig. 3) was synthesized at SmithKline Beecham. Stoichiometric E·Ile-AMP complex was prepared by incubating IleRS with excess [Mg·ATP] and [Ile] followed by gel filtration (Pharmacia Fast Desalt) to remove excess substrates. Adenylated IleRS was stable for several hours when stored on ice (data not shown). Stopped-flow studies, and the resulting data analysis (16Leatherbarrow R.J. Grafit, Version 3.0. Erithacus Software Ltd., Staines, UK1992Google Scholar, 17Barshop B.A. Wrenn R.F. Freiden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (670) Google Scholar, 18Mendes P. Comput. Appl. Biosci. 1993; 9: 563-571PubMed Google Scholar, 19Mendes P. Trends Biochem. Sci. 1997; 22: 361-363Abstract Full Text PDF PubMed Scopus (496) Google Scholar), were performed with an Applied Photophysics SM17MV instrument at 22 °C in 50 mm Tris-HCl, pH 7.9, 10 mm MgCl2 (buffer B) as described (12Pope A.J. Moore K.J. McVey M. Mensah L. Benson N. Osbourne N. Broom N. Brown M.J.B. O’Hanlon P. J. Biol. Chem. 1998; 273: 31691-31701Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Although IleRS (E) from S. aureus possesses 18 tryptophan residues distributed fairly uniformly throughout the sequence (11Chalker A. Ward J. Fosberry A. Hodgson J. Gene (Amst.). 1994; 141: 103-108Crossref PubMed Scopus (22) Google Scholar), examination of the equilibrium enzyme fluorescence of various complexes showed that, with the exception of E·ATP, these differed markedly in their fluorescence yields (Fig. 1). This provided the opportunity to determine the rates and equilibria for their inter-conversion directly by stopped-flow techniques. The intrinsic protein fluorescence intensity is increased by around 7% in the E·Ile binary complex and by around 17% upon the catalytic formation of E·Ile-AMP (Fig. 1). However, enzyme fluorescence was not altered by the presence of >5 mmMg·ATP, Mg·PPi, or Mg·Ap4A (adenosine 5′-tetraphospho-5′-adenosine; data not shown). Interestingly, binding of Ile-ol-AMP (Fig. 10 a) yielded an identical enzyme fluorescence to E·Ile-AMP formed during the reaction cycle, suggesting that the conformation of the enzyme-ligand complex is similar in both cases. The fluorescence change induced by Ile-ol-AMP was used to monitor those interactions that are spectroscopically silent (e.g. Mg·ATP binding, see Fig. 4) using a kinetic competition approach (see below).Figure 4Kinetic competition of Ile-ol-AMP and Mg·ATP binding to IleRS. A, stopped-flow traces obtained from mixing 0.1 μm E with 0.5 μm Ile-ol-AMP plus 0, 2, and 10 mm ATP·Mg.B, dependence of k obs on [ATP] (0.02–15 mm) from experiments such as those shown in A. The solid line is the best fit of the data to Equation 1 yielding K 2 = 2.5 mm.C, kinetic simulation of the predicted fluorescence transients (arbitrary units) for the experiments shown in Ausing Scheme FS1 and the data in Table I.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have characterized the elementary rate and/or equilibrium constants involved in substrate binding, activation, and Ile-ol-AMP binding using stopped-flow according to the minimal mechanism shown in Scheme FS1. The rate and equilibrium constants derived (Table I, Scheme FS1) provide an adequate description of all of the experimental steady-state and transient kinetic data as described below.Table IRate and equilibrium constants for substrate binding, activation and inhibitor binding to S. aureus IleRS · E = IleRSStepRate/equilibrium constantsIle + E ↔ E · Ilek+1 = 2.2 ± 0.5 × 106 m −1 s−1k−1 = 130 ± 50 s−1K1 (=k−1/k+1) = 60 ± 27 μmIle + E · ATP ↔ E · Ile · ATPk+4 = 1.7 ± 0.2 × 106 m −1 s−1k−4 = 3 ± 1.5 s−1K4 (=k−4/k+4) = 1.7 ± 0.9 μmATP + E ↔ E · ATPK2 (=k−2/k+2) = 2.5 ± 0.2 mmATP + E · Ile ↔ E · Ile · ATPK3 (=k−3/k+3) = 70 ± 10 μmE · Ile · ATP ↔ E · Ile-AMP · PP1k+5 = 60 s−1, k−5 = 150 − 200 s−1, K5(=k+5/k−5) ≈ 0.3 − 0.4E · Ile-AMP · PPi ↔ E · Ile-AMP + PP1K6 (= k+6/k−6) = 250 ± 25 μmIle-ol-AMP + E ↔ E · Ile-ol-AMPk+i = 2.4 ± 0.2 × 106 m −1 s−1, k−i = 0.07 ± 0.05 s−1, Ki (=k−i/k+i) ≈ 30 nmThe fluorescence yields (in arbitrary units of nm −1) of the various enzyme intermediates used in the simulations of Scheme FS1 were: E = E · ATP = 1.00; E · Ile = E · Ile · ATP = 1.08; E · Ile-AMP · PPi = E · Ile-AMP = E · Ile-ol-AMP = 1.16 and where the predicted Fobs = [E] + [E · ATP ] + 1.08 ([E · Ile ] + [E · ATP · Ile ]) + 1.16([E · Ile · AMP · PPi] + [E · Ile-AMP ] + [E · Ile-ol-AMP]). Open table in a new tab The fluorescence yields (in arbitrary units of nm −1) of the various enzyme intermediates used in the simulations of Scheme FS1 were: E = E · ATP = 1.00; E · Ile = E · Ile · ATP = 1.08; E · Ile-AMP · PPi = E · Ile-AMP = E · Ile-ol-AMP = 1.16 and where the predicted Fobs = [E] + [E · ATP ] + 1.08 ([E · Ile ] + [E · ATP · Ile ]) + 1.16([E · Ile · AMP · PPi] + [E · Ile-AMP ] + [E · Ile-ol-AMP]). See below 2Equilibrium (dissociation) or steady-state constants for step n are termed K n and Kn ′ for reactions performed in the absence and presence of tRNA, respectively. The rate and equilibrium constants are defined explicitly in Scheme FS1 (Table I). The internal equilibrium constant, K 5, and the external equilibrium constant, K 6, are written in the forward direction. K i (=k −i/k +i) is the equilibrium dissociation constant for inhibitor binding to E. Where elementary rate and equilibrium constants cannot be directly extracted from derivative plots of the observed rate constants,k obs,1 and k obs,2 against concentration, the limiting rate constants and apparent equilibrium constants have the suffix app (e.gk +5,app or K3,app). Thek cat values for the aminoacylation and ATP/PPi exchange reactions arek cat,tr and k cat,ex, respectively. for explanation of the notation used throughout this and accompanying (12Pope A.J. Moore K.J. McVey M. Mensah L. Benson N. Osbourne N. Broom N. Brown M.J.B. O’Hanlon P. J. Biol. Chem. 1998; 273: 31691-31701Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 13Pope A.J. McVey M. Fantom K. Moore K.J. J. Biol. Chem. 1998; 273: 31702-31706Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) papers. The binding of excess Ile-ol-AMP to IleRS was monitored directly by rapid mixing of the enzyme and inhibitor in a stopped-flow apparatus (Fig. 2 A). The observed rate constant, k obs, of the exponential increase in protein fluorescence (amplitude ≈17%) varied linearly with Ile-ol-AMP concentration (Fig. 2 B), consistent with a simple bimolecular reaction (Scheme FS1; k obs =k +i·[Ile-ol-AMP] + k −i). Although k +i = 2.4 ± 0.2 × 106 m −1 s−1(n = 3), the intercept defining k −iwas too small to be determined accurately (k −i < 0.5 s−1) from Fig. 2 B. However, direct measurement of k −i for Ile-ol-AMP via dis-placement experiments yielded a value of ∼0.07 s−1 (12Pope A.J. Moore K.J. McVey M. Mensah L. Benson N. Osbourne N. Broom N. Brown M.J.B. O’Hanlon P. J. Biol. Chem. 1998; 273: 31691-31701Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), defining an overallK i ∼ 30 nm. Similar experiments to those described above but with the substrate l-isoleucine (Ile), also resulted in a single exponential increase in protein fluorescence (Fig. 3 A). The experiment shown in Fig. 3 provided an estimate of k +1 = 3.1 ± 0.3 × 106 m −1·s−1,k -1 = 142 ± 14 s−1 (Fig. 3 B; K 1 = 46 ± 6 μm). Six independent repeat experiments yielded mean (±S.D.) values for each parameter of k +1 = 2.2 ± 0.5 × 106 m −1·s−1,k -1 = 131 ± 50 s−1, and henceK 1 = 60 ± 27 μm. This large error in K 1 limits the reliability of other equilibrium constants in the mechanism, particularlyK 4, which have been estimated, in part, from a thermodynamic linkage argument involving the estimate of K 1 (see below). Nevertheless, these rate and equilibrium constants are consistent with the [Ile] dependence of the fluorescence amplitudes (Fig. 3 C, K 1= 56 ± 10 μm) and provide unambiguous evidence that IleRS can bind Ile (K 1 ≈ 50–100 μm) in the absence of Mg·ATP. The value fork +1 is lower than expected for a diffusion limited binding process (typically >108 m −1 s−1 (37Gutfreund H. Enzymes: Physical Principles. Wiley Interscience, New York1975: 157-161Google Scholar)), although several mechanisms can give rise to such behavior (14Fersht A.R. Enzyme Structure and Mechanism. W. H. Freeman & Co., New York1984Google Scholar, 20Gutfreund H. Kinetics for the Life Sciences. Cambridge University Press, Cambridge, UK1995Crossref Google Scholar, 36Johnson K. Boyer P. The Enzymes. 3rd Ed. 20. Academic Press, London1992: 1-61Google Scholar) and for the purposes of Scheme FS1, Ile binding can be considered an elementary step.E·Ile­ol­AMP ⇌k+i[Ile­ol­AMP]k−i E ⇌k−2k+2[ATP] E·ATP SCHEME 2 The steady-state protein fluorescence intensity of IleRS was not detectably changed upon addition of 5 mmMg·ATP (Fig. 1), a concentration approximately equal to 2× K2 ′ and ∼10-fold higher than the Km, ATP ′ measured in the steady state tRNA aminoacylation reaction (see below, Fig. 9), suggesting that ATP binding was spectroscopically silent. We therefore employed a transient kinetic competition approach (21Moore K.J. Lohman T.M. Biochemistry. 1994; 33: 14565-14578Crossref PubMed Scopus (38) Google Scholar) to characterize the binding of Mg·ATP to E. In these experiments, ATP is pre-mixed in the syringe of the stopped-flow apparatus with a ligand (e.g. Ile-ol-AMP) which is both competitive with ATP (Fig. 10) (12Pope A.J. Moore K.J. McVey M. Mensah L. Benson N. Osbourne N. Broom N. Brown M.J.B. O’Hanlon P. J. Biol. Chem. 1998; 273: 31691-31701Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and which induces a fluorescence change upon binding toE (Figs. 1 and 2). This mixture is then rapidly mixed withE such that the two ligands bind to Esimultaneously and in parallel according to Scheme 2 and the transient kinetics of E·Ile-ol-AMP formation are monitored spectroscopically.The kinetics and/or thermodynamics of ATP binding are inferred from the effect of ATP on the kinetics of Ile-ol-AMP binding (21Moore K.J. Lohman T.M. Biochemistry. 1994; 33: 14565-14578Crossref PubMed Scopus (38) Google Scholar). A series of kinetic competition experiments were performed by mixing 0.1 μm E with a sample of 0.5 μmIle-ol-AMP plus varying concentrations of Mg·ATP. Mixing Einto Ile-ol-AMP alone yielded, as expected (Fig. 2), a single exponential increase in protein fluorescence (k obs ≈ 2.5 s−1, amplitude ≈16%, Fig. 4 A). When Mg·ATP and Ile-ol-AMP were mixed with E, k obs for the formation of E·Ile-ol-AMP decreased with increasing [ATP] (Fig. 4), as expected for the competition between the two ligands where the binding of the spectroscopically silent ligand (ATP) is rapidly reversible relative to the rate of Ile-ol-AMP binding (21Moore K.J. Lohman T.M. Biochemistry. 1994; 33: 14565-14578Crossref PubMed Scopus (38) Google Scholar). Analysis of the data according to Equation 1 yields an estimate forK 2 of ∼2.5 ± 0.2 mm(mean ± S.D., n = 3) and an estimate of k −i (∼0.1 s−1) consistent with that determined directly (Fig. 2), as shown, kobs=k+i[Ile­ol­AMP]1+[ATP]K2+k−i(Eq. 1) As predicted, the estimate of K 2 was independent of the concentration of Ile-ol-AMP used (definingk -2 > 10 s−1; see also Fig. 9), and was similar to that obtained from the activation of the aminoacylation reaction by ATP (see below, 2.7 mm; Table III). These data therefore provide clear evidence that ATP can bind toE in the absence of the co-substrate, Ile (and vice versa, see Fig. 3), such that a minimal mechanism must include random order of addition of both substrates. Simulations of the experiment shown in Fig. 4 A by numerical integration techniques (using Scheme FS1, Table I) provide an adequate description of the experimental data (compare Fig. 4, A and C).Table IIISteady-state parameters for tRNA aminoacylation and PPi/ATP exchange by S. aureus IleRS measured at 22 °C, pH 7.9 (see “Materials and Methods”)Kinetic parametertRNA aminoacylationPPi/ATP exchangeKm ,Ile(μm)5 ± 310 ± 2Km ,ATP (μm)240 ± 18ND3-aND, not done.Km ,tRNA(μm)<0.1NA3-bNA, not applicable.kcat (s−1)0.35 ± 0.0518.0 ± 2kcat/Km ,Ile(m −1 s−1)1.8 × 1051.8 × 105Data represent the mean (± S.E. or range) of 2–3 independent experiments3-a ND, not done.3-b NA, not applicable. Open table in a new tab Data represent the mean (± S.E. or range) of 2–3 independent experiments Rapid mixing of IleRS with mixtures of Ile and Mg·ATP to initiate the amino acid activation reaction resulted in a 16–17% increase in enzyme fluorescence (cf. Fig. 1). Although the time course of the change in species concentration predicted by Scheme FS1 is complex (see below), due to the particular combination of fluorescence yields associated with each of the intermediates, the observed fluorescence transients could, under most conditions, be described by the sum of two exponentials (e.g Fig. 5 A). The fast phase of the transient (observed rate constant k obs,1) had an [Ile] dependence consistent with that observed previously (Fig. 3) and an equivalent transient was observed in the absence of ATP. Furthermore, this rate constant was invariant when IleRS was mixed into varying ATP concentrations in the presence of a fixed concentration of Ile (Fig. 5 B, displayed on a logarithmic scale for clarity). The first phase therefore corresponds to the binding of Ile to the fraction of E that is nucleotide-free (at the concentration of ATP used relative to K 2) to formE·Ile. As described below, the binding of ATP to formE·ATP leads to a reduction in the dissociation rate constant for Ile binding but no significant change in the association rate constant. If k obs,1 arose from the binding of Ile to E·ATP, k obs,1 would be predicted to be lower than the equivalent rate constant observed in the absence of added ATP, in contrast to that observed experimentally. The slower transient observed in these mixing experiments,k obs,2 (ΔF ≈ 7%), showed a hyperbolic dependence in either [ATP] (Fig. 5 C) or [Ile] (data not shown, Table II), most likely due to the first-order catalytic formation of theE·Ile-AMP intermediate since the fluorescence yield of E·Ile-AMP is about 7% greater than that of E·Ile (or E·ATP, E·Ile·ATP) alone (Fig. 1). The [ATP] dependence of k obs,2was analyzed according to Equation 2 ([L] = [Mg·ATP]) to obtain estimates of the maximal observed rate constant,k max = k +5,app +k -5,app, the apparent K d of the binding step,K 3,app and the apparent reverse rate constant for the first order step, k -5,app. We emphasize the apparent nature of these terms as opposed to the elementary nature of the rate and equilibrium constants reported in Table I 2 and derived as described, kobs=k+5,app·[L]K3,app+[L]+k−5,app(Eq. 2) The value of k -5,app was too low to be determined reliably in these experiments, but had a value of <2 s−1 such that k max (=k +5,app + k -5,app) approximates to k +5,app. According to Scheme FS1, the apparent irreversibility of the chemical cleavage step (k -5,app < 2 s−1) is a consequence of the weak binding of PPi, the rapid and thermodynamically favorable release of which from theE·Ile-AMP·PPi complex makes theobserved reverse rate negligible.Table IIDependence of the rate constant for IleRS · Ile-AMP · PPi formation (kobs, 2) on substrate concentration when IleRS was rapidly mixed with Ile and ATP in experiments analogous to those shown in Fig. 5CFixed substrateVariable substrateK4,app(μm)K3,app(μm)Observed k+5,app + k−5,app≈ k+5,app (s−1)Simulated k+5,app + k−5,app ≈ k+5,app(s−1)ATP · Mg 1 mmIsoleucine16 ± 753 ± 657 2 mm15 ± 554 ± 558Isoleucine 15 μmMg · ATP86 ± 728 ± 0.524 30 μm72 ± 1442 ± 1.841 60 μm71 ± 1557 ± 2.658Derivative plots (kobs, 2 versus[substrate]) were fitted to Equation 2 to obtain estimates of the limiting rate constant at high concentration, kmax = k+5,app + k−5,app and K3,appor K4,app (see text for details). Open table in a new tab Derivative plots (kobs, 2 versus[substrate]) were fitted to Equation 2 to obtain estimates of the limiting rate constant at high concentration, kmax = k+5,app + k−5,app and K3,appor K4,app (see text for details). To determine if the maximal apparent rate constant observed at high [ATP] in Fig. 5 C (57 s−1) was the elementary rate constant for E·Ile-AMP·PPi formation, we conducted a series of experiments in which the concentration dependence of k obs,2 in either Ile or ATP was measured in the presence of fixed concentrations of the other substrate (Table II). The maximal observed rate constant,k max, was dependent upon [Ile], reaching a limiting value near 55 s−1 at high concentrations of both ATP and Ile (Table II). This rate constant likely reflects the true elementary rate constant for E·Ile-AMP·PPiformation which was confirmed in a number of other experiments conducted at saturating concentrations of Ile and ATP (not shown). Kinetic simulations of these experiments (Scheme FS1, Table I) yielded fluorescence transients similar to those observed experimentally (compare Fig. 5, A and D) and furthermore, predicted accurately the apparent maximal rate constants fork obs,2 at different [Ile] (Table II). The apparent equilibrium constants for ATP and Ile (K 3,app = 75 μm and K 4,app = 15 μm, respectively; Table II) only approximate to the true equilibrium constants,K 3 and K 4, when the pseudo first-order association and first-order dissociation rate constants are much larger than the rate constant for the subsequent (signal generating) chemical step. For ATP binding toE·Ile, kinetic simulations were able to predict the observed fluorescence transients using a value of K 3 (70 μm) approximately equal to the K 3,app value from Table II (75 μm). However, similar kinetic simulations indicate that the K 4,app value for Ile binding toE·ATP (15 μm) is an overestimate of the trueK 4 (1.7 ± 0.9 μm, Table I). Indeed, experiments described below suggest that the dissociation of Ile from E·Ile·ATP (k -4 ∼ 3 ± 1.5 s−1) occurs more slowly than the rate of the chemical cleavage step (∼60 s−1). As such, the non-equivalence of the best estimate of K 4 and the K 4,app for Ile activation of the chemistry step (Table II) is to be expected. Finally, based on reasonably reliable estimates for three of the four equilibrium constants in Scheme FS1 defining the formation of the E·ATP·Ile complex, thermodynamic linkage with associated error propagation (primarily in K 1, see above), suggestsK 4 = 1.7 ± 0.9 μm. Despite this slight uncertainty, the major conclusion is that the binding of either substrate to E reduces the K d for the formation of the ternary E·ATP·Ile complex by >10-fold compared with the K d obtained for either substrate binding to free E (Figs. 3 and 4). Although Table II predicts that Ile binds more tightly to E·ATP than to the free enzyme, there is no information concerning the rate constantsk +4 and k -4. Initial experiments to monitor the association kinetics of Ile withE·ATP (prior to the chemical step) proved difficult to interpret since, at low [Ile], k obs,1(defining the formation of the ternary E·ATP·Ile complex) had a similar rate constant and amplitude tok obs,2 (defining the catalytic formation of E·Ile-AMP·PPi). In an attempt to kinetically resolve the bimolecular Ile binding step from the first order catalytic step, we conducted experiments at high [Ile] (up to 300 μm) although this necessarily led to rapid transients and associated uncertainty (Fig. 6 A). The apparent observed rate constant increased approximately linearly with [Ile] (Fig. 6 B), yielding an intercept of 22 ± 13 s−1 and a slope of 1.6 × 106 m −1 s−1. These values only represent k -4 and k +4when the bimolecular formation of E·ATP·Ile occurs much more rapidly than the decay of the ternary complex (at ∼60 s−1). However, the predicted species distribution (based on Scheme FS1, Table I) suggests that both processes are likely to be kinetically linked below 150 μm Ile (Fig. 6 D). Only the data between 150 and 250 μm Ile (which yieldedk obs ∼ 450 s−1; close to the upper limit for reliable measurements on our apparatus) could be used to estimate the elementary rate constants (k +4 ∼ 1.6 × 106 m −1 s−1) and, therefore, we can estimate only k -4 <10 s−1. This value is, as we noted above, significantly less than the estimate of the rate of the chemistry step (about 60 s−1) such that rapid equilibrium assumptions associated with Ile binding toE·ATP are invalid. Despite the difficulty in obtaining a reliable estimate for k -4, and henceK 4, from either Table I or Fig. 6 or by thermodynamic linkage with other more well defined equilibrium constants we obtain an estimate of K 4 = 1.7 ± 0.9 μm and hence k -4 ∼ 3 ± 1.5 s−1 from an estimate of k +4. Kinetic simulation of the experiments (Fig. 6 A, Scheme FS1, Table I, k -4 ∼ 3 ± 1.5 s−1) provided an adequate description of the experimental transients (Fig. 6 C) and of the apparent rate constant for the chemistry step at different [Ile] (Table II). We believe the combined data justifies the estimate of k -4 and henceK 4 with appropriate caveats regarding the uncertainty associated with these value. Binding of ATP to free E was sufficiently weak (K 2 = 2.5 mm) to be expected to be a rapid equilibrium on the stopped-flow time scale, an assumption we have incorporated into simulations of Scheme FS1 (k +2 = 107 m −1 s−1,k -2 = 2.5 × 104s−1, e.g. Fig. 6 D). As such, we would expect no difference in the kinetics of E·Ile-AMP formation when the E·ATP complex was mixed with Ile and when both substrates were mixed with E simultaneously. To test this hypothesis (Fig. 7), a fixed concentration of the pre-bound substrate was maintained in both stopped-flow syringes, so that any pre-established equilibria were not perturbed upon subsequent mixing with the second substrate. As expected, the stopped-flow time course obtained was identical whether or not ATP was pre-mixed with E (Fig. 7 A, note that the traces have been offset for clarity). In contrast, the fluorescence amplitude observed following pre-binding of Ile toE reflects the difference in fluorescence intensity betweenE·Ile and E·Ile-AMP (Fig. 1). Kinetic simulation of the experiments in Fig. 7 A (Scheme FS1, Table I) provided a reasonable description of the experimental data (Fig. 7 B) with exponential rate constants (46 s−1) comparable to those observed experimentally (40–44 s−1). Since theE·Ile-AMP complex is relatively stable (rate constant for decay of E·Ile-AMP < 3 × 10−4s−1; data not shown), it could be isola" @default.
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