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• W2094348523 abstract "A steady state kinetic investigation of the Pi activation of 5-phospho-d-ribosyl α-1-diphosphate synthase from Escherichia coli suggests that Pi can bind randomly to the enzyme either before or after an ordered addition of free Mg2+ and substrates. Unsaturation with ribose 5-phosphate increased the apparent cooperativity of Pi activation. At unsaturating Pi concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with Pi directs the subsequent ordered binding of Mg2+ and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pibinds to the enzyme last. The random mechanism for Pibinding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying Pi at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing Piconcentrations. Results from ADP inhibition of Piactivation suggest that these effectors compete for binding to a common regulatory site. A steady state kinetic investigation of the Pi activation of 5-phospho-d-ribosyl α-1-diphosphate synthase from Escherichia coli suggests that Pi can bind randomly to the enzyme either before or after an ordered addition of free Mg2+ and substrates. Unsaturation with ribose 5-phosphate increased the apparent cooperativity of Pi activation. At unsaturating Pi concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with Pi directs the subsequent ordered binding of Mg2+ and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pibinds to the enzyme last. The random mechanism for Pibinding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying Pi at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing Piconcentrations. Results from ADP inhibition of Piactivation suggest that these effectors compete for binding to a common regulatory site. 5-phospho-d-ribosyl α-1-diphosphate ribose 5-phosphate (+)-1-α,2-α,3-α-trihydroxy-4-β-cyclopentanemethanol 5-phosphate α,β-methylene ATP α,β-methylene MgATP The enzyme 5-phospho-d-ribosyl α-1-diphosphate (PRPP)1 synthase (EC 2.7.6.1) catalyzes the reaction MgATP + Rib-5-P → AMP + PRPP. PRPP is a precursor of purine, pyrimidine and pyridine nucleotides and the amino acids histidine and tryptophan (1Jensen K.F. Munch-Petersen A. Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganisms. Academic Press, London1983: 1-25Google Scholar, 2Hove-Jensen B. J. Bacteriol. 1988; 170: 1148-1152Crossref PubMed Google Scholar, 3Hove-Jensen B. Mol. Microbiol. 1989; 3: 1487-1492Crossref PubMed Scopus (48) Google Scholar). In addition, PRPP is a precursor of methanopterin in Methanosarcina thermophila (4White R.H. Biochemistry. 1996; 35: 3447-3456Crossref PubMed Scopus (42) Google Scholar) and polyprenylphosphate-pentoses in Mycobacteria (5Scherman M.S. Kalbe-Bournonville L. Bush D. Xin Y. Deng L. McNeil M. J. Biol. Chem. 1996; 271: 29652-29658Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The PRPP synthase reaction proceeds by attack of the 1-hydroxyl of Rib-5-P on the β-phosphoryl of ATP resulting in the transfer of the β,γ-diphosphoryl moiety of ATP to Rib-5-P (6Khorana H.G. Fernandes J.F. Kornberg A. J. Biol. Chem. 1958; 230: 941-948Abstract Full Text PDF PubMed Google Scholar, 7Miller Jr., G.A. Rosenzweig S. Switzer R.L. Arch. Biochem. Biophys. 1975; 171: 732-736Crossref PubMed Scopus (24) Google Scholar). Mg2+ions are required to form the actual substrate MgATP and as an activator of the enzyme (8Switzer R.L. J. Biol. Chem. 1969; 244: 2854-2863Abstract Full Text PDF PubMed Google Scholar, 9Switzer R.L. J. Biol. Chem. 1971; 246: 2447-2458Abstract Full Text PDF PubMed Google Scholar, 10Arnvig K. Hove-Jensen B. Switzer R.L. Eur. J. Biochem. 1990; 192: 195-200Crossref PubMed Scopus (49) Google Scholar, 11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar, 12Roth D.G. Shelton E. Deuel T.F. J. Biol. Chem. 1974; 249: 291-296Abstract Full Text PDF PubMed Google Scholar, 13Fox I.H. Kelley W.N. J. Biol. Chem. 1972; 247: 2126-2131Abstract Full Text PDF PubMed Google Scholar). PRPP synthases from Salmonella typhimurium (8Switzer R.L. J. Biol. Chem. 1969; 244: 2854-2863Abstract Full Text PDF PubMed Google Scholar, 14Gibson K.J. Schubert K.R. Switzer R.L. J. Biol. Chem. 1982; 257: 2391-2396Abstract Full Text PDF PubMed Google Scholar, 15Switzer R.L. Sogin D.C. J. Biol. Chem. 1973; 248: 1063-1073Abstract Full Text PDF PubMed Google Scholar), Escherichia coli (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar, 16Hove-Jensen B. Harlow K.W. King C.J. Switzer R.L. J. Biol. Chem. 1986; 261: 6765-6771Abstract Full Text PDF PubMed Google Scholar),Bacillus subtilis (10Arnvig K. Hove-Jensen B. Switzer R.L. Eur. J. Biochem. 1990; 192: 195-200Crossref PubMed Scopus (49) Google Scholar), human (17Becker M.A. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar), and rat (18Sonoda T. Ishizuka T. Ishijima S. Kita K. Ahmad I. Tatibana M. Biochim. Biophys. Acta. 1998; 1387: 32-40Crossref PubMed Scopus (9) Google Scholar) possess an absolute requirement for Pi as an activator and are subject to inhibition by ADP and for B. subtilis and mammalian enzymes also by GDP, which binds to a specific allosteric site. In addition, ADP competes with MgATP for binding to the active site. A second class of PRPP synthases, so far only found in plants, is independent of Pi for activity (19Krath B.N. Eriksen T.A. Poulsen T.S. Hove-Jensen B. Biochim. Biophys. Acta. 1999; 1430: 403-408Crossref PubMed Scopus (30) Google Scholar, 20Krath B.N. Hove-Jensen B. Plant Physiol. 1999; 119: 497-506Crossref PubMed Scopus (34) Google Scholar). The enzymes from S. typhimurium and E. coli share identical primary sequences except for two conservative replacements (16Hove-Jensen B. Harlow K.W. King C.J. Switzer R.L. J. Biol. Chem. 1986; 261: 6765-6771Abstract Full Text PDF PubMed Google Scholar, 21Bower S.G. Harlow K.W. Switzer R.L. Hove-Jensen B. J. Biol. Chem. 1989; 264: 10287-10291Abstract Full Text PDF PubMed Google Scholar, 22Bower S.G. Hove-Jensen B. Switzer R.L. J. Bacteriol. 1988; 170: 3243-3248Crossref PubMed Google Scholar), which is also reflected in their similar, if not identical, enzymological properties. We have previously shown that Mg2+, MgATP, and Rib-5-P bind in that order to E. coli PRPP synthase by a steady state ordered mechanism and allosteric inhibition by ADP appeared competitive against activation by free Mg2+ at subsaturating Rib-5-P concentrations (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar). Inhibition by ADP and GDP was also shown to increase the half-saturation constant for Mg2+ activation of rat PRPP synthases I and II (18Sonoda T. Ishizuka T. Ishijima S. Kita K. Ahmad I. Tatibana M. Biochim. Biophys. Acta. 1998; 1387: 32-40Crossref PubMed Scopus (9) Google Scholar). From previous analysis of the enzymes fromS. typhimurium (14Gibson K.J. Schubert K.R. Switzer R.L. J. Biol. Chem. 1982; 257: 2391-2396Abstract Full Text PDF PubMed Google Scholar, 15Switzer R.L. Sogin D.C. J. Biol. Chem. 1973; 248: 1063-1073Abstract Full Text PDF PubMed Google Scholar) and E. coli (16Hove-Jensen B. Harlow K.W. King C.J. Switzer R.L. J. Biol. Chem. 1986; 261: 6765-6771Abstract Full Text PDF PubMed Google Scholar), it was found that ADP appears to bind to the allosteric site only in the presence of Rib-5-P. As a consequence, the interaction of PRPP synthase with the allosteric inhibitor ADP appears to involve ADP binding to the allosteric site of the enzyme prior to Mg2+ binding as well as to the enzyme in complex with Mg2+ and substrates. From analysis of the hydrodynamic properties of S. typhimurium PRPP synthase, it was found that Pimaintains the oligomeric structure of the enzyme (23Schubert K.R. Switzer R.L. Shelton E. J. Biol. Chem. 1975; 250: 7492-7500Abstract Full Text PDF PubMed Google Scholar) and that removal of Pi results in irreversible loss of activity (8Switzer R.L. J. Biol. Chem. 1969; 244: 2854-2863Abstract Full Text PDF PubMed Google Scholar, 16Hove-Jensen B. Harlow K.W. King C.J. Switzer R.L. J. Biol. Chem. 1986; 261: 6765-6771Abstract Full Text PDF PubMed Google Scholar). The role of Pi in the steady state kinetics of PRPP synthase has not previously been analyzed in detail. Indirect evidence that allosteric Pi activation and ADP inhibition occur by competition for binding to the same site has been presented. The analysis of mutant forms of human PRPP synthase I that have a reduced sensitivity to allosteric inhibition by ADP and GDP revealed a concomitant increase in affinity for Pi (17Becker M.A. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar). The inhibitor 4-amino-8-(β-d-ribofuranosylamino)pyrimido[5,4-d]pyrimidine appears to bind at the allosteric site of both human PRPP synthases I and II, and the concentration of the inhibitor needed for half-maximal inhibition increased with increasing Pi concentration (24Fry D.W. Becker M.A. Switzer R.L. Mol. Pharmacol. 1995; 47: 810-815PubMed Google Scholar). The recent structure of the B. subtilis PRPP synthase in complex with ADP or sulfate ions revealed a hexameric arrangement. The structures indicated that the allosteric site defined by three subunits is the target for binding of both ADP and Pi, the latter being represented by a sulfate ion (25Eriksen T.A. Kadziola A. Bentsen A.K. Harlow K.W. Larsen S. Nat. Struct. Biol. 2000; 7: 303-308Crossref PubMed Scopus (77) Google Scholar). Together these observations suggest a more subtle mechanism behind Pi activation apart from maintaining the structure. To gain a more detailed understanding of the regulation of the PRPP synthase, we have analyzed the steady state kinetics of Pi activation of the E. colienzyme and suggest a complete model for the interaction of PRPP synthase with all of its known ligands. ATP was obtained from Roche Molecular Biochemicals, mATP and Rib-5-P were obtained from Sigma, and cRib-5-P was a gift from R. J. Parry (Rice University, Houston, TX) (26Parry R.J. Burns M.R. Skae P.N. Hoyt J.C. Pal B. Bioorg. Med. Chem. 1996; 4: 1077-1088Crossref PubMed Scopus (13) Google Scholar). [8-14C]ADP was from Amersham Pharmacia Biotech. E. coli PRPP synthase was purified as described previously (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar, 27Willemoës M. Nilsson D. Hove-Jensen B. Biochemistry. 1996; 35: 8181-8186Crossref PubMed Scopus (17) Google Scholar) and had a specific activity of approximately 150 μmol min−1 mg−1 when assayed in the presence of 2 mm ATP, 5 mmRib-5-P, 5 mm MgCl2 as described below. The protein concentration was determined by the bicinchoninic acid procedure (28Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18349) Google Scholar) with reagents provided by Pierce and with bovine serum albumin as a standard. The 32P transfer assay was performed at 37 °C in 55 mmtriethanolamine, pH 8.0, as described previously (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar, 27Willemoës M. Nilsson D. Hove-Jensen B. Biochemistry. 1996; 35: 8181-8186Crossref PubMed Scopus (17) Google Scholar), except that enzyme was diluted in 2 mm ATP, 10 mmMgCl2, 50 mm triethanolamine, pH 8.0, bovine serum albumin (1 mg ml−1). The concentrations of divalent metal ion and nucleotide complexes and free divalent metal ions were calculated as described previously (9Switzer R.L. J. Biol. Chem. 1971; 246: 2447-2458Abstract Full Text PDF PubMed Google Scholar, 11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar, 15Switzer R.L. Sogin D.C. J. Biol. Chem. 1973; 248: 1063-1073Abstract Full Text PDF PubMed Google Scholar). Unless otherwise noted, the free Mg2+ concentration varied within a saturating level of 3–5 mm, because of the varying Piconcentrations. The MgATP concentration was maintained at 2 mm. The nucleotides ADP and mATP was calculated to be more than 90% complexed with Mg2+. The binding of Ca2+ by ATP was neglected when calculating the free Ca2+ concentration, because Mg2+ was present in at least 15-fold excess. The stability constant used for the calculation of the Ca2+ complex with Pi was 0.03 mm−1 (29Smith R.M. Alberty R.A. J. Amer. Chem. Soc. 1956; 78: 2376-2380Crossref Scopus (163) Google Scholar). The concentration of Pi, Rib-5-P and inhibitors varied as indicated. Results of initial velocity experiments were analyzed by fitting data by nonlinear regression to the appropriate Equations Equation 1, Equation 2, Equation 3, Equation 4, Equation 5, Equation 6 using the computer program UltraFit (BioSoft, version 3.01). The standard errors for kinetic parameters presented are those calculated by the program. Equation 1 is the Michaelis-Menten equation for hyperbolic substrate saturation kinetics, Equation 2 is the Hill equation for cooperative substrate saturation kinetics, Equation 3 is a general equation for nonhyperbolic saturation kinetics (30Ferdinand W. Biochem. J. 1966; 98: 278-283Crossref PubMed Scopus (130) Google Scholar, 31Neet K.E. Methods Enzymol. 1980; 64: 139-192Crossref PubMed Scopus (112) Google Scholar), Equation 4 applies to noncompetitive inhibition, and Equations 5 and 6 apply to nonlinear competitive and nonlinear noncompetitive inhibition, respectively, and where the effect of the inhibitor on S 0.5 is caused by successive binding of two molecules of inhibitor at different sites on the enzyme. Equations Equation 4, Equation 5, Equation 6 apply to cooperative substrate saturation kinetics, where n is not affected by the presence of inhibitor (32Zhang R. Villeret V. Lipscomb W.N. Fromm H.J. Biochemistry. 1996; 35: 3038-3043Crossref PubMed Scopus (47) Google Scholar). v=VappS/(Km+S)Equation 1 v=VappSn/(S0.5n+Sn)Equation 2 v=(aS+bS2)/(1+cS+dS2)Equation 3 v=VappSn/(S0.5n[1+I/Kis]+Sn[1+I/Kii])Equation 4 v=VappSn/(S0.5n[1+I/Kis1+I2/Kis1Kis2]+Sn)Equation 5 v=VappSn/(S0.5n[1+I/Kis1+I2/Kis1Kis2]+Sn[1+(I/Kii)])Equation 6 where v is the initial velocity;V app is the apparent maximal velocity;S is the concentration of the varied substrate or activator;K m is the apparent Michaelis-Menten constant forS; S 0.5 is the half-saturation concentration for S; n is the apparent Hill-coefficient for S; a, b,c, and d are complex functions of rate constants and the concentration of nonvaried substrates and as such have no physical meaning; K is,K is1, andK is2 are inhibition constants for the effect on S 0.5, where a suffix, 1 or 2, onK is refers to the two different binding constants for nonlinear inhibition; and K ii is the inhibitor constant for the effect on V app. All velocities are in μmol min−1 mg−1. Ligand binding was performed as described previously (33Ormö M. Sjöberg B.M. Anal. Biochem. 1990; 189: 138-141Crossref PubMed Scopus (51) Google Scholar, 34Lundegaard C. Jensen K.F. Biochemistry. 1999; 38: 3327-3334Crossref PubMed Scopus (37) Google Scholar). PRPP synthase (4.5 nmol) was incubated at pH 8.2 in 150 μl of 50 mm Pi, 25 mm Tris-HCl, 5 mm MgCl2 and varying concentrations of ADP (0.2 nCi of [8-14C]ADP per incubation). When present the Rib-5-P concentration was 2 mm. Each incubation was transferred to a Millipore Ultrafree-MC centrifugal filter unit and equilibrated to 25 °C and centrifuged for 5–10 min at 5000 × g in a thermostated microcentrifuge (OLE DICH Instruments, Copenhagen, Denmark). Samples (30 μl) from the incubation prior to centrifugation (total ligand) and from the eluent after centrifugation (free ligand) was withdrawn, and radioactivity was quantitated with a Packard 2000 liquid scintillation analyzer. The ADP binding data were analyzed by fitting to Equation 7 or 8 for data obtained in the absence or presence of Rib-5-P, respectively. Equation 7 applies to simple hyperbolic binding, and Equation 8 is a two-site binding model with one site, the allosteric site, showing cooperative binding. N=AmaxL/(KA+L)Equation 7 N=AmaxL/(KA+L)+BmaxLnb/(KBnb+Lnb)Equation 8 where N is mol ADP bound per mol monomer;A max and B max are the numbers of active sites and allosteric sites per monomer of enzyme (34,000 kDa), respectively; L is the unbound ADP concentration; K A and K Bare the half-saturation constants for the active site and the allosteric site, respectively; and nb is the Hill coefficient for binding to the allosteric site. PRPP synthase from E. coli is normally stored and diluted in the presence of 50 mm Pito maintain stability (16Hove-Jensen B. Harlow K.W. King C.J. Switzer R.L. J. Biol. Chem. 1986; 261: 6765-6771Abstract Full Text PDF PubMed Google Scholar). Therefore, to study Piactivation it was necessary to find a condition where the enzyme was stable in the absence of Pi. With 2 mm MgATP or more the enzyme was found to be fully stable upon dilution and subsequent incubation at 37 °C. Because concentrations of free Mg2+ that were kinetically subsaturating, in combination with 2 mm MgATP, fully stabilized the enzyme, it was possible to study the influence of Mg2+ and Rib-5-P, but not MgATP, on the activation of PRPP synthase by Pi. Saturation with free Mg2+ resulted in nearly hyperbolic activation of PRPP synthase by Pi, whereas cooperative activation by Pi was observed at unsaturating free Mg2+ concentrations (Fig.1 A). Apparently, also theS 0.5 for Pi increased when the free Mg2+ concentration was lowered to 14 μm (Fig.1 A). Mg2+ activation appeared hyperbolic at Pi concentrations of 5 mm and above but was clearly cooperative at 1.5 mm Pi (Fig.1 B). However, the concentration for half-saturation with free Mg2+ changed by less than a factor of two over the entire range of Pi concentrations (Fig. 1 B). The apparent cooperativity of Pi activation increased when the Rib-5-P concentration was lowered to 0.5 mm and showed a 2-fold decrease in S 0.5 for Picompared with the results obtained at 5 mm Rib-5-P (Fig.2 A). At 0.5 mmRib-5-P, beginning inhibition by Pi concentrations exceeding 25 mm was observed, probably because of competitive binding to the Rib-5-P binding site. Apparently, the saturation of PRPP synthase with Rib-5-P was sensitive to the Pi concentration because increasing Rib-5-P concentrations induced partial substrate inhibition that was relieved by 50 mm Pi (Fig. 2 B).Figure 1Activation of PRPP synthase by Pior Mg2+. Assays were performed as described under “Experimental Procedures.” A, Pi varied as indicated in the presence of the indicated concentrations of Mg2+. ▵, data were fitted to Equation 2;V app = 130 ± 3,S 0.5 = 3.1 ± 0.2 mm,n = 1.3 ± 0.1. ○, data were fitted to Equation2; V app = 73 ± 1,S 0.5 = 2.6 ± 0.1 mm,n = 2.0 ± 0.2. ▿, data were fitted to Equation2; V app = 39.1 ± 0.8,S 0.5 = 8.5 ± 0.3 mm,n = 2.0 ± 0.1. B, Mg2+varied as indicated in the presence of the indicated concentrations of Pi. ▵, data were fitted to Equation 1;V app = 129 ± 4, K m = 41 ± 4 μm. ○, data were fitted to Equation 1;V app = 103 ± 6, K m = 59 ± 10 μm. ▿, data were fitted to Equation 2;V app = 55 ± 2, S 0.5= 69 ± 5 μm, n = 2.1 ± 0.2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Saturation of PRPP synthase by Pior Rib-5-P. Assays were performed as described under “Experimental Procedures.” A, Pi varied as indicated in the presence of the indicated concentrations of Rib-5-P. ▵, data were fitted to Equation 2; V app = 150 ± 6, S 0.5 = 2.6 ± 0.3 mm, n = 1.2 ± 0.1. ○, data were fitted to Equation 2; V app = 129 ± 2,S 0.5 = 1.65 ± 0.05 mm,n = 2.1 ± 0.1. B, Rib-5-P varied as indicated in the presence of the indicated concentrations of Pi. ▵, data were fitted to Equation 1;V app = 157 ± 2, K m = 0.19 ± 0.01 mm. ○, ▿, and ⋄, data were fitted to Equation 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Divalent calcium, MgmATP, and cRib-5-P have been shown to competitively inhibit the binding of Mg2+, MgATP, and Rib-5-P, respectively (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar). Results from experiments where Pi was varied in the presence of different fixed concentrations of inhibitor at saturating or nonsaturating Rib-5-P concentrations are presented in Table I. All three inhibitors exhibited linear noncompetitive inhibition of Pi binding regardless of the Rib-5-P concentration. At the tested concentrations of inhibitor and Rib-5-P neither Ca2+, MgmATP, nor cRib-5-P induced inhibition by increasing Pi concentrations.Table IMode of inhibition of Pi activation by inhibitors of PRPP synthaseInhibitorMode of inhibition[Rib-5-P] 1-aThe Pi concentration varied from 0 to 50 mm, except with 0.5 mmRib-5-P, where the Pi concentration varied from 0 to 25 mm.K isK iiK is1K is2mmμmμmμmμmCa2+ b,cnoncompetitive5.032 ± 647 ± 4Ca2+ b,cnoncompetitive0.553 ± 10110 ± 6MgmATP 1-dThe MgmATP concentration varied from 0 to 1.0 mm.noncompetitive5.0229 ± 65230 ± 19MgmATP 1-dThe MgmATP concentration varied from 0 to 1.0 mm.noncompetitive0.5790 ± 279560 ± 34cRib-5-P c,enoncompetitive1.01180 ± 325716 ± 33ADP 1-fConditions were as described in the legend to Fig. 3 A.nonlinear5.018 ± 2111 ± 24competitiveADP 1-gConditions were as described in the legend to Fig. 3 B.nonlinear0.5334 ± 2815 ± 340 ± 14noncompetitiveInhibition constants were determined as described under “Experimental Procedures.”b The Ca2+ concentration varied from 0 to 0.2 mm.c The free Mg2+ concentration was maintained at 1.2 mm.e The cRib-5-P concentration varied from 0 to 1.0 mm.1-a The Pi concentration varied from 0 to 50 mm, except with 0.5 mmRib-5-P, where the Pi concentration varied from 0 to 25 mm.1-d The MgmATP concentration varied from 0 to 1.0 mm.1-f Conditions were as described in the legend to Fig. 3 A.1-g Conditions were as described in the legend to Fig. 3 B. Open table in a new tab Inhibition constants were determined as described under “Experimental Procedures.” b The Ca2+ concentration varied from 0 to 0.2 mm. c The free Mg2+ concentration was maintained at 1.2 mm. e The cRib-5-P concentration varied from 0 to 1.0 mm. When Pi was varied at either 0.5 mm or 5 mm Rib-5-P, the presence of increasing fixed concentrations of ADP resulted in a pronounced nonlinear effect onS 0.5 for Pi (Fig.3 and Table I). The effect of ADP on bothS 0.5 and V app for saturation of the enzyme by Pi in the presence of 0.5 mm Rib-5-P could readily be determined when the data were fitted to Equation 5 (Fig. 3 B and Table I). Data from ADP inhibition of Pi saturation at 5 mm Rib-5-P were fitted as nonlinear competitive inhibition because no effect of ADP on V app could be estimated for the data within the range of Pi concentrations available to us (Fig.3 A and Table I). Increasing the concentration of Pi beyond 50 mm in the assay incubation results in the rapid formation of a MgPiprecipitate. An observed cooperativity in binding of MgmATP to PRPP synthase (14Gibson K.J. Schubert K.R. Switzer R.L. J. Biol. Chem. 1982; 257: 2391-2396Abstract Full Text PDF PubMed Google Scholar) at 0 °C was shown to be an effect of the temperature at which the experiment was performed, because it is absent at 25 °C (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar). To investigate whether the high degree of cooperativity associated with ADP binding to the allosteric site previously determined (14Gibson K.J. Schubert K.R. Switzer R.L. J. Biol. Chem. 1982; 257: 2391-2396Abstract Full Text PDF PubMed Google Scholar) would also be influenced by temperature, we performed ADP binding experiments at 25 °C. However, the extent of cooperativity in ADP binding to the allosteric site at 25 °C (Fig.4) was not significantly different from that determined previously at 0 °C (14Gibson K.J. Schubert K.R. Switzer R.L. J. Biol. Chem. 1982; 257: 2391-2396Abstract Full Text PDF PubMed Google Scholar). Steady state kinetics and ligand binding studies of the S. typhimurium and E. coli PRPP synthases have identified the mechanism for Mg2+, MgATP, and Rib-5-P binding to the enzyme as occurring in that order (8Switzer R.L. J. Biol. Chem. 1969; 244: 2854-2863Abstract Full Text PDF PubMed Google Scholar, 9Switzer R.L. J. Biol. Chem. 1971; 246: 2447-2458Abstract Full Text PDF PubMed Google Scholar, 11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar). Allosteric binding of ADP has been shown to occur either at conditions where the enzyme is fully saturated with Mg2+ and substrates (14Gibson K.J. Schubert K.R. Switzer R.L. J. Biol. Chem. 1982; 257: 2391-2396Abstract Full Text PDF PubMed Google Scholar, 15Switzer R.L. Sogin D.C. J. Biol. Chem. 1973; 248: 1063-1073Abstract Full Text PDF PubMed Google Scholar, 16Hove-Jensen B. Harlow K.W. King C.J. Switzer R.L. J. Biol. Chem. 1986; 261: 6765-6771Abstract Full Text PDF PubMed Google Scholar) or prior to binding of Mg2+ and substrates as revealed by cooperative competitive ADP inhibition of Mg2+ activation (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar). By comparing MgmATP and ADP inhibition of Pi activation at 0.5 mm Rib-5-P (Table I) the K ii for ADP is likely to represent binding to the active site in competition with MgATP. The nonlinear effect of ADP on S 0.5 for Pi described by K is1 andK is2 is observed both at 0.5 and 5 mm Rib-5-P. This nonlinear effect of ADP onS 0.5 for Pi is sufficient to explain the inhibition by ADP at 5 mm Rib-5-P. Although aK ii for ADP at 5 mm Rib-5-P would have been expected by comparing with MgmATP inhibition under similar conditions, this could not be extracted from the data. The nonlinear effect of ADP on S 0.5 for Pi, (TableI) suggests that ADP competes with Pi for binding to the regulatory site. However, because neitherK is1 norK is2 is comparable withK is for MgmATP under similar conditions, we hesitate to further assign an exact physical meaning forK is1 andK is2. It appears that ADP and Pi compete for binding to the same enzyme form in a manner largely independent of the Rib-5-P concentration. On the basis of the previous data from ADP inhibition and ligand binding studies mentioned above, we suggest that ADP and Pi compete for binding to the allosteric site of the enzyme either prior to or after binding of Mg2+ and substrates. The results of Fig. 2 shows the characteristics of a steady state random mechanism (30Ferdinand W. Biochem. J. 1966; 98: 278-283Crossref PubMed Scopus (130) Google Scholar, 31Neet K.E. Methods Enzymol. 1980; 64: 139-192Crossref PubMed Scopus (112) Google Scholar, 35Wells B.D. Stewart T.A. Fisher J.R. J. Theor. Biol. 1976; 60: 209-221Crossref PubMed Scopus (8) Google Scholar) with a preferred pathway in which Pi binds to the enzyme prior to Rib-5-P. The kinetic pattern shown in Fig. 2 is likely to result from a difference in the magnitude of the rate constants of otherwise similar kinetic equilibria for a random binding of Pi and Rib-5-P. Because substrate inhibition by Rib-5-P is only partial, it is unlikely to result from formation of a dead end complex. The apparent noncompetitive inhibition of Pi activation observed by Ca2+, MgmATP, or cRib-5-P (Table I) suggests two mechanisms that both would agree with the results of Fig. 2. Either substrates can bind fully random to the enzyme with respect to Pi (i.e. before or after binding of Pi) by a steady state random mechanism, or an ordered binding of Mg2+ and MgATP is proceeded by a random binding of Rib-5-P and Pi. In favor of a fully random mechanism is the absence of inhibition at increasing Pi concentrations induced by the presence of inhibitor. This would be observed to the extent that obligatory binding of the inhibitor to the enzyme occurs prior to Pi and if downstream binding of substrates and Pi also occurs to the enzyme inhibitor complex (36Cleland W.W. Methods Enzymol. 1979; 63: 500-513Crossref PubMed Scopus (148) Google Scholar). Both Ca2+ (15Switzer R.L. Sogin D.C. J. Biol. Chem. 1973; 248: 1063-1073Abstract Full Text PDF PubMed Google Scholar) and MgmATP (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar) induce substrate inhibition by Rib-5-P in agreement with the ordered binding mechanism where Rib-5-P binds to the enzyme last. A random mechanism where Mg2+ and substrates bind to PRPP synthase either before or after Pibinding therefore appears most consistent with the results presented here and those previously obtained as mentioned above. In Scheme FS1 an outline of the interaction of PRPP synthase with substrates, activators, and ADP is suggested. The allosteric effectors Pi and ADP compete for binding to the allosteric site of either the free enzyme or enzyme in complex with Mg2+ and substrates. To explain the data of Fig. 2, we have made the assumption that a fast pathway and a slow pathway exist from the free enzyme to the catalytic complex. Furthermore, it is assumed that the binding order of Mg2+, MgATP, and Rib-5-P is conserved whether Pi is bound to the enzyme or not. The noncompetitive inhibition of Pi activation by Ca2+, MgmATP, and Rib-5-P is also consistent with the mechanism in Scheme FS1. According to Scheme FS1 the Mg2+ activation of PRPP synthase should resemble the saturation of the enzyme with Rib-5-P under similar conditions. Accordingly, the results of Fig. 1 A where unsaturation of PRPP synthase with Mg2+ yields cooperative Pi activation can be explained by a preferred pathway in a random mechanism. However, unlike the saturation with Rib-5-P at low Pi concentrations (Fig. 2 B), the Mg2+ activation at 1.5 mm Pi is cooperative (Fig. 1 B), and no inhibition by increasing Mg2+ concentrations is observed. Because apparently no cooperativity is associated with Ca2+ inhibition of Pi activation, it may suggest that the apparent cooperativity of Mg2+ activation at low Pi is not due to homotropic site-site interactions. The apparent cooperativity of Mg2+ activation in the presence of 1.5 mm Pi (Fig. 1 B) may be interpreted in terms of the apparent increase in S 0.5 for Pi at low Mg2+ (Fig. 1 A). If the affinity of the enzyme for Pi binding via the slow pathway is relatively higher, it may favor this pathway over the fast pathway at low Mg2+ concentrations. We realize that Scheme FS1 must somehow be a simplification of the actual overall mechanism for PRPP synthase. One consistent observation that is not explained by Scheme FS1 is the high degree of cooperativity for binding of ADP to the allosteric site (Fig. 4) with Hill coefficients between 3 and 4.4 when determined in the presence of 50 mmPi (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar, 14Gibson K.J. Schubert K.R. Switzer R.L. J. Biol. Chem. 1982; 257: 2391-2396Abstract Full Text PDF PubMed Google Scholar). Our results from ADP inhibition of Pi activation at 0.5 mm and 5 mmRib-5-P can be explained without including any cooperativity associated with the ADP inhibition. It seems controversial that ADP and Pi can only bind to the allosteric site of either free enzyme or enzyme complexed with Mg2+ and substrates. However, at present there seems to be no experimental evidence supporting the possibility that ADP can bind to the intermediates of Scheme FS1 other than those indicated, and because ADP and Piapparently compete for binding to the same form(s) of the enzyme, this should be true for Pi as well. Apart from explaining the observed competition between binding of Mg2+ and ADP (11Willemoës M. Hove-Jensen B. Biochemistry. 1997; 36: 5078-5083Crossref PubMed Scopus (24) Google Scholar,18Sonoda T. Ishizuka T. Ishijima S. Kita K. Ahmad I. Tatibana M. Biochim. Biophys. Acta. 1998; 1387: 32-40Crossref PubMed Scopus (9) Google Scholar) as a simple consequence of a random mechanism, Scheme FS1 also allows for an equilibrium between active and inactive forms of the enzyme that can be shifted by allosteric effectors and apparently by specific amino acid changes, as suggested from analysis of human mutant enzymes (17Becker M.A. Smith P.R. Taylor W. Mustafi R. Switzer R.L. J. Clin. Invest. 1995; 96: 2133-2141Crossref PubMed Scopus (75) Google Scholar). The recent solving of the crystal structure of B. subtilisPRPP synthase (25Eriksen T.A. Kadziola A. Bentsen A.K. Harlow K.W. Larsen S. Nat. Struct. Biol. 2000; 7: 303-308Crossref PubMed Scopus (77) Google Scholar) seems very promising for our attempts to understand the mechanism behind the allosteric regulation of the enzyme. The kinetic analysis presented here suggests what complexes can be expected to be formed between the enzyme and its ligands. When the structural details of more complexes are known other than those likely to represent free enzyme complexed with ADP and Pi, we may address specific questions about the regulatory mechanism in SchemeFS1. The mechanism in Scheme FS1 also addresses the question of actual substrate and activator concentrations under physiological conditions. Because the response of the PRPP synthase to substrates, activators, and ADP seems very dependent on the concentration of Rib-5-P and Pi, it may suggest that the mechanism proposed in Scheme FS1can play a regulatory role in determining the rate of PRPP synthesis in response to changes in metabolite concentrations. This is the topic of work currently in progress in our laboratories. We thank Jørgen Andresen for excellent technical assistance. K. Frank Jensen and Robert L. Switzer are acknowledged for valuable discussions." @default.
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• W2094348523 title "Steady State Kinetic Model for the Binding of Substrates and Allosteric Effectors to Escherichia coliPhosphoribosyl-diphosphate Synthase" @default.
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