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• W2080962613 abstract "Cooperativity is extensively used by enzymes, particularly those acting at key metabolic branch points, to “fine tune” catalysis. Thus, cooperativity and enzyme catalysis are intimately linked, yet their linkage is poorly understood. Here we show that negative cooperativity in the rate-determining step in the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex is an outcome of redistribution of a “rate-promoting” conformational pre-equilibrium. An array of biophysical and biochemical studies indicates that non-catalytic but conserved residues directly regulate the redistribution. Furthermore, factors such as ligands and temperature, individually or in concert, also strongly influence the redistribution. As a consequence, these factors also exert their influence on catalysis by profoundly influencing the pre-equilibrium facilitated dynamics of communication between multienzyme components. Our observations suggest a mode of cooperativity in the E1 component that is consistent with the dynamical hypothesis shown to satisfactorily explain cooperativity in many well studied enzymes. The results point to the likely existence of multiple modes of communication between subunits when the entire class of thiamin diphosphate-dependent enzymes is considered. Cooperativity is extensively used by enzymes, particularly those acting at key metabolic branch points, to “fine tune” catalysis. Thus, cooperativity and enzyme catalysis are intimately linked, yet their linkage is poorly understood. Here we show that negative cooperativity in the rate-determining step in the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex is an outcome of redistribution of a “rate-promoting” conformational pre-equilibrium. An array of biophysical and biochemical studies indicates that non-catalytic but conserved residues directly regulate the redistribution. Furthermore, factors such as ligands and temperature, individually or in concert, also strongly influence the redistribution. As a consequence, these factors also exert their influence on catalysis by profoundly influencing the pre-equilibrium facilitated dynamics of communication between multienzyme components. Our observations suggest a mode of cooperativity in the E1 component that is consistent with the dynamical hypothesis shown to satisfactorily explain cooperativity in many well studied enzymes. The results point to the likely existence of multiple modes of communication between subunits when the entire class of thiamin diphosphate-dependent enzymes is considered. Allostery in proteins emanates from a redistribution of the conformational ensemble (1.Hilser V.J. Thompson E.B. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8311-8315Crossref PubMed Scopus (328) Google Scholar, 2.Cooper A. Dryden D.T.F. Eur. Biophys. J. 1984; 11: 103-109Crossref PubMed Scopus (555) Google Scholar). The generally accepted notion is that binding of a ligand to one site can affect the other through a propagated change in the protein shape; however, it has been shown that allosteric communication could also exist in the absence of such physical linkage (1.Hilser V.J. Thompson E.B. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 8311-8315Crossref PubMed Scopus (328) Google Scholar). In the latter scenario, communication between structurally separated active sites is thermodynamic in nature. Cooperativity, a special case of allostery, is closely associated with ligand-induced conformational dynamics, which may provide free energy of allosteric coupling via entropic effects (2.Cooper A. Dryden D.T.F. Eur. Biophys. J. 1984; 11: 103-109Crossref PubMed Scopus (555) Google Scholar). Well characterized thiamin diphosphate (ThDP) 2The abbreviations used are: ThDPthiamin diphosphatePLThDPC2α-phosphonolactyl-ThDPMAPmethyl acetylphosphonate sodium saltE1ecE1 component of the E. coli pyruvate dehydrogenase multienzyme complexE2ecE. coli dihydrolipoamide acetyltransferase componentE3ecE. coli dihydrolipoamide dehydrogenase componentE551C-TFAtrifluoroacetonyl group introduced at E551C-substituted cysteineless E1ecITCisothermal titration calorimetry. -dependent enzymes are homodimers (α2), homotetramers (α4), or heterotetramers (α2β2) and have two or four active sites formed at subunit interfaces. Recent investigations to find out whether these active sites, such as in many dimeric and multimeric enzymes, communicate with each other have yielded divergent and contradictory hypotheses (3.Seifert F. Golbik R. Brauer J. Lilie H. Schröder-Tittmann K. Hinze E. Korotchkina L.G. Patel M.S. Tittmann K. Biochemistry. 2006; 45: 12775-12785Crossref PubMed Scopus (49) Google Scholar, 4.Frank R.A. Titman C.M. Pratap J.V. Luisi B.F. Perham R.N. Science. 2004; 306: 872-876Crossref PubMed Scopus (148) Google Scholar, 5.Li J. Machius M. Chuang J.L. Wynn R.M. Chuang D.T. J. Biol. Chem. 2007; 282: 11904-11913Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). These diverse but compelling results suggest that there may not be a single unified mechanism explaining cooperativity in all ThDP-dependent enzymes; rather, multiple modes may be utilized to achieve the catalytic goals suited to the particular pathway in which these enzymes participate (6.Jordan F. Nemeria N.S. Sergienko E.A. Acc. Chem. Res. 2005; 38: 755-763Crossref PubMed Scopus (28) Google Scholar). thiamin diphosphate C2α-phosphonolactyl-ThDP methyl acetylphosphonate sodium salt E1 component of the E. coli pyruvate dehydrogenase multienzyme complex E. coli dihydrolipoamide acetyltransferase component E. coli dihydrolipoamide dehydrogenase component trifluoroacetonyl group introduced at E551C-substituted cysteineless E1ec isothermal titration calorimetry. The E1 component (E1ec) of the Escherichia coli pyruvate dehydrogenase multienzyme complex is an α2 homodimer and consists of two active centers at the monomer interfaces, each binding one ThDP and one Mg2+ ion (7.Arjunan P. Nemeria N. Brunskill A. Chandrasekhar K. Sax M. Yan Y. Jordan F. Guest J.R. Furey W. Biochemistry. 2002; 41: 5213-5221Crossref PubMed Scopus (125) Google Scholar). The x-ray structures of the apo-E1ec (7.Arjunan P. Nemeria N. Brunskill A. Chandrasekhar K. Sax M. Yan Y. Jordan F. Guest J.R. Furey W. Biochemistry. 2002; 41: 5213-5221Crossref PubMed Scopus (125) Google Scholar), E1ec with ThDP bound (7.Arjunan P. Nemeria N. Brunskill A. Chandrasekhar K. Sax M. Yan Y. Jordan F. Guest J.R. Furey W. Biochemistry. 2002; 41: 5213-5221Crossref PubMed Scopus (125) Google Scholar), or E1ec with C2α-phosphonolactyl-ThDP bound (PLThDP; a stable analogue of the predecarboxylation covalent intermediate of ThDP formed with the substrate analogue methyl acetylphosphonate (MAP)) (8.Arjunan P. Sax M. Brunskill A. Chandrasekhar K. Nemeria N. Zhang S. Jordan F. Furey W. J. Biol. Chem. 2006; 281: 15296-15303Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and of apo-E1ec and E1ec variants complexed with ThDP and PLThDP (9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) revealed no structural inequivalence of the active centers and hence provided no hint as to how the two sites communicate structurally. The active center region of E1ec is highly dynamic, exhibiting ligand- and temperature-dependent conformational equilibrium involving the dynamic inner (encompassing residues 401–413) and outer loops (encompassing residues 541–557) from different subunits of an α2 dimer along with an always ordered helical segment (residues 525–535) (8.Arjunan P. Sax M. Brunskill A. Chandrasekhar K. Nemeria N. Zhang S. Jordan F. Furey W. J. Biol. Chem. 2006; 281: 15296-15303Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Using an array of biophysical and biochemical methods, we showed that this conformational equilibrium is also present in the unliganded enzyme (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar). Such a “pre-equilibrium” is an intrinsic attribute of key functional proteins and has been shown to have important catalytic roles in many enzymes. Consistent with this idea, the pre-equilibrium in E1ec was shown to be critical for many of its catalytic functions starting from predecarboxylation events and culminating in the transfer of the acetyl moiety to the E2ec component (9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Moreover, the pre-equilibrium was shown to “energetically promote” the covalent addition of substrate to the enzyme bound ThDP by decreasing the activation energy and was found to be rate-determining (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar). Pre-steady state analysis of the rate-determining step in E1ec revealed that it could be resolved into at least two phases: a fast phase followed by a slower one. This kinetic response suggested that one set of sites reacts rapidly, whereas the other set reacts slowly, a hallmark of negative cooperative behavior in which two active sites act asymmetrically, indicating intersubunit communication. A conformational pre-equilibrium is also an important feature of many enzymes exhibiting cooperativity in its action. Therefore, the pre-equilibrium in E1ec may also be an important modulator of the observed cooperative behavior. Although allosteric communication between distant sites is fundamental to the catalytic functions of enzymes, allosteric communication linked to catalytic turnover is poorly understood (11.Stetefeld J. Jenny M. Burkhard P. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13688-13693Crossref PubMed Scopus (31) Google Scholar). In this study, we provide experimental evidence that the dynamics of both active center loops exhibit synchronous pre-equilibrium that occurs on a time scale similar to that of the rate-limiting catalytic step, and is essential for catalysis. Using an array of methods, we further show that the cooperativity detected in the E1ec rate-determining step is dynamically modulated and is the outcome of a redistribution of rate-promoting pre-equilibrium in an ensemble in response to interplay of temperature and ligand binding. The E. coli strain JRG 3456 deficient in native E1ec gene was transformed with pGS878 plasmid containing the aceE gene encoding the E1ec and was used for overexpression and site-directed mutagenesis. The pET-22b(+)-1-lip E2 vector transformed in E. coli BL21 (DE3) cells was used for overexpression of 1-lip E2, a construct with a single lipoyl domain, instead of three lipoyl domains in the wild-type E2ec, but shown to be very similar in biochemical properties to the latter but more suitable to mechanistic studies. Activity was measured with either 1-lip E2 or 3-lipoyl domain E2ec (the latter was obtained from the National BioResource Project (Japan)) and E3ec as described earlier (12.Nemeria N. Volkov A. Brown A. Yi J. Zipper L. Guest J.R. Jordan F. Biochemistry. 1998; 37: 911-922Crossref PubMed Scopus (30) Google Scholar). The mass ratio of E1ec·E2ec·E3ec complex was 1:5:5. The procedures for expression, purification, and activity measurements of E1ec and its singly substituted variants were described previously (13.Yi J. Nemeria N. McNally A. Jordan F. Machado R.S. Guest J.R. J. Biol. Chem. 1996; 271: 33192-33200Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14.Nemeria N. Yan Y. Zhang Z. Brown A.M. Arjunan P. Furey W. Guest J.R. Jordan F. J. Biol. Chem. 2001; 276: 45969-45978Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 15.Park Y.H. Wei W. Zhou L. Nemeria N. Jordan F. Biochemistry. 2004; 43: 14037-14046Crossref PubMed Scopus (20) Google Scholar). The following primers (substitutions underlined) were used for construction of singly substituted E1ec variants: N548A, 5′-AGTACACCCCGGCGGACCGCGAGCAGGTTGC-3′; E551A, 5′-GCAGGACCGCGCGCAGGTTGCTTACTATAAAG-3′; E551C, 5′-CAGTATACCCCGCAGGATCGCTGTCAGGTGGCG-3′;R550A, 5′-CAGTACACCCCGCAGGACGCCGAGCAGGTTGC-3′; D549A, 5′-CAGTACACCCCGCAGGCCCGCGAGCAGGTTGC-3′. CD spectroscopy was used to determine the dissociation constant (Kd(PLThDP)) for binding of substrate analog MAP to E1ec and loop variants and was essentially similar to the method described earlier (9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Stopped-flow CD spectra were recorded on the π*-180 CDF spectrometer (Applied Photophysics, Leatherhead, UK). Temperature was adjusted to the desired value with an Rtϵ 7 Thermo NESLAB temperature controller. A slit width of 2 mm and path length of 10 mm were used. Reactions were monitored for the indicated times, and data were analyzed and prepared using Pro-K Global analysis software (Applied Photophysics) and Sigma Plot (Systat Software, Inc.), respectively. The PLThDP formation traces were fitted to a double exponential equation (y = A0 − A1exp(−k1t) − A2exp(−k2t)). The temperature dependence of pre-steady state kinetic data was analyzed by the Arrhenius equation, lnk=lnA-(EaRT)(Eq. 1) where Ea is the activation energy, R is the gas constant, A is a pre-exponential factor, and T is temperature. Fluorescence quenching experiments were performed to determine the binding affinity of ThDP (Kd(ThDP)) as described previously (14.Nemeria N. Yan Y. Zhang Z. Brown A.M. Arjunan P. Furey W. Guest J.R. Jordan F. J. Biol. Chem. 2001; 276: 45969-45978Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The time dependence of reductive acetylation of independently expressed lipoyl domain by the E1ec component or by outer loop E1ec variants was monitored using a 4800 Plus matrix-assisted laser desorption ionization time-of-flight/time-of-flight analyzer (Applied Biosystems, Foster City, CA). The procedure was essentially similar to the one described previously (9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The changes in relative intensities were fit to first-order decay (y = y0 + A1e−x/t) to derive the apparent rate constant, k′r (k′r = 1/t), for reductive acetylation. This procedure was described in detail previously (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar). The proximal location of residues Asp549, Gln548 located on the outer loop of one subunit, and Asn404 from the inner loop from the second subunit within hydrogen bonding distance (Fig. 1) and their conservation in homodimeric pyruvate dehydrogenase multienzyme complexes and 2-oxoacid dehydrogenase complexes (supplemental Fig. S1) suggests that these residues might have important role(s) in enzyme function (9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The crystal structure of E1ec with the intermediate analogue PLThDP bound does not suggest direct involvement of Asp549 or Asn404 in predecarboxylation steps due to their large distance (at least in the ordered conformation) from the active center ThDP. However, distal residues have been shown to play important roles in catalysis and regulation by influencing the rate-promoting dynamic processes (16.Agarwal P.K. Billeter S.R. Rajagopalan P.T. Benkovic S.J. Hammes-Schiffer S. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 2794-2799Crossref PubMed Scopus (408) Google Scholar, 17.Rajagopalan P.T. Lutz S. Benkovic S.J. Biochemistry. 2002; 41: 12618-12628Crossref PubMed Scopus (162) Google Scholar, 18.Tousignant A. Pelletier J.N. Chem. Biol. 2004; 11: 1037-1042Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). To test this hypothesis with E1ec, we created variants with alanine substitutions at these residues. The E1ec-specific activity (via reduction by the enamine of 2,6-dichlorophenolindophenol or DCPIP) for D549A and N404A was only modestly reduced by factors of 5 and 2, respectively, whereas the activity of the entire complex (NADH production after reconstitution with E2ec and E3ec) was reduced ∼260- and ∼56-fold, respectively (Fig. 2A and supplemental Table S1). This precipitous drop in overall complex activity as compared with DCPIP activity is an indication that the substitutions greatly impair intercomponent communication. Furthermore, the time-dependent reductive acetylation of lipoyl domain by D549A and N404A proceeded at a greatly reduced rate. Although E1ec completes this reaction in <30 s (our minimum reaction time for quenching manually mixed components), for the D549A and N404A variants, this reaction was incomplete even after 30 min of incubation, since the unacetylated lipoyl domain could still be detected in the reaction medium (Fig. 2B). The apparent rate constant for reductive acetylation (k′r) (the rate of transfer of acetyl group from E1ec to the lipoyl moiety on E2ec) was significantly lower as compared with that for E1ec. These results are in line with our previous results on other inner loop variants (9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and support a generalized conclusion drawn with respect to ThDP-dependent enzymes, that loop disorder to order transition, manifested as pre-equilibrium in E1ec, also confers specificity of lipoyl domain recognition during transfer of acetyl group to the E2ec component (8.Arjunan P. Sax M. Brunskill A. Chandrasekhar K. Nemeria N. Zhang S. Jordan F. Furey W. J. Biol. Chem. 2006; 281: 15296-15303Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 19.Wynn R.M. Kato M. Machius M. Chuang J.L. Li J. Tomchick D.R. Chuang D.T. Structure. 2004; 12: 2185-2196Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20.Nemeria N. Arjunan P. Brunskill A. Sheibani F. Wei W. Yan Y. Zhang S. Jordan F. Furey W. Biochemistry. 2002; 41: 15459-15467Crossref PubMed Scopus (31) Google Scholar, 21.Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (482) Google Scholar, 22.Jones D.D. Stott K.M. Reche P.A. Perham R.N. J. Mol. Biol. 2001; 305: 49-60Crossref PubMed Scopus (21) Google Scholar). This observation also supports the recent hypothesis (3.Seifert F. Golbik R. Brauer J. Lilie H. Schröder-Tittmann K. Hinze E. Korotchkina L.G. Patel M.S. Tittmann K. Biochemistry. 2006; 45: 12775-12785Crossref PubMed Scopus (49) Google Scholar) that for those ThDP enzymes that are part of multienzyme complexes (unlike the enzymes that are not part of such complexes), the active center dynamics also serves to support substrate channeling or, in the case of E1ec, the transfer of acetyl group to E2ec.FIGURE 2Biochemical signatures of pre-equilibrium disruption. A, activities and kinetic parameters of outer loop variants. The E1-specific assay used DCPIP reduction monitored at 600 nm (1 unit of activity = 1 μmol of DCPIP reduced/min/mg of E1ec). For overall complex activity (pyruvate:NAD+ oxidoreductase activity), the production of NADH was monitored at 340 nm (1 unit of activity = 1 μmol of NAD+ reduced/min/mg of E1ec). Detailed kinetic parameters of variants are summarized in supplemental Table S1. B, determination of dissociation constant for thiamin diphosphate (Kd(ThDP)) in E1ec and its variants. C, time-dependent reductive acetylation of lipoyl domain by D549A and N404A. Inner loop variants that resulted in impaired pre-equilibrium (9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) are also shown for comparison. The apparent pseudo-first order rate constant (k′r) is also shown. D–F, formation of “carboligase” side reaction products acetolactate and acetoin on the E1ec, D549A, and N404A, respectively ((S)-acetoin gives a positive band with λmax near 280 nm, whereas (R)-acetolactate gives rise to a negative band with λmax at 301–302 nm) (21.Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (482) Google Scholar). The variants were incubated for 16 h at 4 °C, and protein was removed in each case with the help of a Centricon YM-30 (Millipore) unit. The carboligation product ratio (acetolactate to acetoin, ([AL]/[AC]), obtained from NMR) for each reaction is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) That the observed effects on kinetics emanate from disruption of interaction between the dynamic regions from two subunits is further supported by the fact that substitution of charged residues in the immediate vicinity of Asp549 and Gln548 (e.g. R550A and E551A) that are not involved in hydrogen bonding with the inner loop did not result in significant reduction in activities (supplemental Table S1). Fluorescence quenching experiments with ThDP yielded similar values of Kd(ThDP) for E1ec, D549A, and N404A (Fig. 2C), indicating that binding of coenzyme (and hence the active center) was unaffected by the substitutions. Therefore, the observed effects on kinetics are probably a result of disruption of interaction between the loops. Moreover, the unusual “carboligation” profile, resulting from impaired sequestering of active site chemistry from solvent (9.Kale S. Arjunan P. Furey W. Jordan F. J. Biol. Chem. 2007; 282: 28106-28116Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), is also indicative of disruption of loop closure, due to disruption of interaction between the loops over the active center in loop-substituted variants (Fig. 2, D–F). Because substitutions on the inner loop invariably affected predecarboxylation steps on E1ec, we determined the ability of D549A and N404A variants to form PLThDP from MAP. Titration of the D549A and N404A with MAP shows small increases in the Kd(PLThDP) (the apparent PLThDP dissociation constant) as compared with E1ec (Fig. 3). Interestingly, both variants exhibited signal saturation at approximately half the CDmax, compared with the CD signal saturated with PLThDP at 300–305 nm observed with E1ec. This suggests that PLThDP formation proceeds in only half of the available sites. Thus, disruption of a dynamic intersubunit interaction among E1ec subunits results in half-of-the-sites reactivity, an extreme form of negative cooperativity. Interestingly, half-of-the-sites reactivity was not present with respect to ThDP (data not shown); this is in contrast to observations on other E1ec variants that displayed half-of-the-sites reactivity for both PLThDP and ThDP (23.Nemeria N. Tittmann K. Joseph E. Zhou L. Vazquez-Coll M.B. Arjunan P. Hübner G. Furey W. Jordan F. J. Biol. Chem. 2005; 280: 21473-21482Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These results suggest that the effects of substitutions are limited solely to substrate turnover. In order to determine whether half-of-the-sites reactivity induced as a result of obliterating intersubunit/interloop interaction is a consequence of altered mobility of the outer loop, we first investigated whether the outer loop (bearing residues Asp549 and Gln548), like the inner loop, exhibits rate-promoting pre-equilibrium and whether pre-equilibrium occurs on catalytic time scales and is thus important for catalysis. Using 19F NMR methodology we utilized earlier (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar) for qualitative and quantitative characterization of inner loop dynamics, we ascertained that the outer loop also exhibits “open-closed” conformational pre-equilibrium (Fig. 4A). The line shape simulations of the 19F NMR spectra of the E551C-TFA (trifluoroacetonyl group introduced at E551C-substituted cysteineless E1ec) (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar) (supplemental Table S2) at different temperatures, within the experimental error range, yielded an exchange rate constant of <1 s−1. In order of magnitude, this value is similar to the observed kcat of the E551C-TFA (kcat = 0.74 s−1 at 30 °C) and kex (kex = kAB + kBA) for the inner loop (also <1 s−1) (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar), suggesting synchronicity and quantitative correlation of outer and inner loop dynamics with catalysis in E1ec. Therefore, we conclude that the dynamics of the two active center loops are concerted and represent a rate-limiting catalytic step or a synchronous rate-promoting pre-equilibrium. Moreover, as observed with the inner loop (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar), Lorentzian deconvolution of the E551C-TFA 19F NMR spectra at different temperatures also revealed that the outer loop open-closed population transition with respect to temperature is not linear; instead, there is a step deviation (Ttran) of pre-equilibrium in favor of the open conformation above 25 °C (Fig. 4B). It has been suggested that ThDP-dependent enzymes are only catalytically active in the “closed” conformation (24.Sundström M. Lindqvist Y. Schneider G. FEBS Lett. 1992; 313: 229-231Crossref PubMed Scopus (67) Google Scholar), whereas the open conformation is a binding-competent conformation as we had observed earlier on E1ec (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar). Therefore, E1ec undergoes a large scale “binding activation transition” at 25 °C. This was also observed in the inner loop populations and is significant because it is also apparent in the variation of kcat with temperature for E1ec (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar). The latter observation indicates that synchronous loop dynamics remains a rate-determining factor (influences kcat) in E1ec catalysis at all temperatures tested. Our earlier thermodynamic studies showed (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar) E1ec to exhibit hallmark features of a protein in which ligand binding is coupled to a conformational change and the thermodynamic signatures of a pre-equilibrium. Briefly, the MAP-binding isotherm to E1ec above (25 °C) revealed a marked initial response in which the heat released per mole of injected MAP increases before declining to zero as the enzyme is saturated with MAP. This suggests that binding of the initial MAP ligands (and presumably substrate pyruvate) occur to thermodynamically distinct sites from the subsequent binding sites, implying interaction between binding sites. The parameters derived revealed that the apparent negative cooperativity at higher temperature is entropically (dynamically) driven. The D549A and N404A variants could not be analyzed by ITC due to low heats. Thus, to determine whether the outer loop substitutions and their effects on cooperativity are caused by disruption of pre-equilibrium, we determined the temperature dependence of kfast and kslow. As with the reaction of MAP with E1ec (see below), kfast increased while kslow decreased with increasing temperature; however, the temperature dependence was once more nonlinear, resulting in nonlinear Arrhenius plots (supplemental Fig. S2A). Nonlinear Arrhenius plots have been attributed to 1) a change in a rate-limiting step, 2) conformational changes in the enzyme, or 3) a change in the specific heat of the reactant. In the present case, since we had shown that the rate-limiting step does not change with temperature (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1158-1163Crossref PubMed Scopus (39) Google Scholar), the observed nonlinearity supports pre-equilibrium and the associated changes in specific heat capacity, as observed in ITC data on E1ec. In fact, a convex Arrhenius plot is characteristic of enzyme-catalyzed reactions involving two or more competing enzymatic forms, each dominating in a different temperature range (25.Massey V. Curti B. Ganther H. J. Biol. Chem. 1966; 241: 2347-2357Abstract Full Text PDF PubMed Google Scholar, 26.Truhlar D. Kohen A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 848-851Crossref PubMed Scopus (132) Google Scholar). A convex Arrhenius plot entails Ea (and hence ΔH*, the enthalpy of activation) decreasing with increasing temperature, concomitant with increasing frequency of rate-promoting process (in the case of E1ec, the rate-limiting pre-equilibrium). Importantly, the discontinuity in kfast and kslow, occurs at ∼25 °C (the “activation transition” temperature observed above), further lowering the Ea required for the kfast (covalent addition of substrate) and kslow (redistribution of pre-equilibrium) processes. This is consistent with ITC (10.Kale S. Ulas G. Song J. Brudvig G.W. Furey W. Jordan F. Proc. Natl. Acad. Sci. U.S.A. 2008;" @default.
• W2080962613 created "2016-06-24" @default.
• W2080962613 creator A5008511786 @default.
• W2080962613 creator A5009378425 @default.
• W2080962613 date "2009-11-01" @default.
• W2080962613 modified "2023-09-26" @default.
• W2080962613 title "Conformational Ensemble Modulates Cooperativity in the Rate-determining Catalytic Step in the E1 Component of the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex" @default.
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