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J. Biol. Chem., Vol. 275, Issue 45, 35408-35412, November 10, 2000

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Steady State Kinetic Model for the Binding of Substrates and Allosteric Effectors to Escherichia coli Phosphoribosyl-diphosphate Synthase^*

Martin Willemoës^§, Bjarne Hove-Jensen^¶, and Sine Larsen

From the  Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark and the ^¶ Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen, Denmark

Received for publication, July 18, 2000, and in revised form, August 11, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A steady state kinetic investigation of the P_i activation of 5-phospho-D-ribosyl -1-diphosphate synthase from Escherichia coli suggests that P_i 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 P_i activation. At unsaturating P_i concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with P_i 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 P_i binds to the enzyme last. The random mechanism for P_i binding was further supported by studies with competitive inhibitors of Mg²⁺, MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying P_i at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing P_i concentrations. Results from ADP inhibition of P_i activation suggest that these effectors compete for binding to a common regulatory site.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The enzyme 5-phospho-D-ribosyl -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 (1-3). In addition, PRPP is a precursor of methanopterin in Methanosarcina thermophila (4) and polyprenylphosphate-pentoses in Mycobacteria (5). The PRPP synthase reaction proceeds by attack of the 1-hydroxyl of Rib-5-P on the -phosphoryl of ATP resulting in the transfer of the ,-diphosphoryl moiety of ATP to Rib-5-P (6, 7). Mg²⁺ ions are required to form the actual substrate MgATP and as an activator of the enzyme (8-13). PRPP synthases from Salmonella typhimurium (8, 14, 15), Escherichia coli (11, 16), Bacillus subtilis (10), human (17), and rat (18) possess an absolute requirement for P_i 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 P_i for activity (19, 20).

The enzymes from S. typhimurium and E. coli share identical primary sequences except for two conservative replacements (16, 21, 22), 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 (11). Inhibition by ADP and GDP was also shown to increase the half-saturation constant for Mg²⁺ activation of rat PRPP synthases I and II (18). From previous analysis of the enzymes from S. typhimurium (14, 15) and E. coli (16), 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 P_i maintains the oligomeric structure of the enzyme (23) and that removal of P_i results in irreversible loss of activity (8, 16). The role of P_i in the steady state kinetics of PRPP synthase has not previously been analyzed in detail.

Indirect evidence that allosteric P_i 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 P_i (17). The inhibitor 4-amino-8-(-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 P_i concentration (24). 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 P_i, the latter being represented by a sulfate ion (25). Together these observations suggest a more subtle mechanism behind P_i 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 P_i activation of the E. coli enzyme and suggest a complete model for the interaction of PRPP synthase with all of its known ligands.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Enzyme Purification-- 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) (26). [8-¹⁴C]ADP was from Amersham Pharmacia Biotech. E. coli PRPP synthase was purified as described previously (11, 27) and had a specific activity of approximately 150 µmol min¹ mg¹ when assayed in the presence of 2 mM ATP, 5 mM Rib-5-P, 5 mM MgCl₂ as described below. The protein concentration was determined by the bicinchoninic acid procedure (28) with reagents provided by Pierce and with bovine serum albumin as a standard.

Assay of PRPP Synthase Activity-- The ³²P transfer assay was performed at 37 °C in 55 mM triethanolamine, pH 8.0, as described previously (11, 27), except that enzyme was diluted in 2 mM ATP, 10 mM MgCl₂, 50 mM triethanolamine, pH 8.0, bovine serum albumin (1 mg ml¹). The concentrations of divalent metal ion and nucleotide complexes and free divalent metal ions were calculated as described previously (9, 11, 15). Unless otherwise noted, the free Mg²⁺ concentration varied within a saturating level of 3-5 mM, because of the varying P_i concentrations. 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 P_i was 0.03 mM¹ (29). The concentration of P_i, Rib-5-P and inhibitors varied as indicated.

Analysis of Enzyme Kinetic Data-- Results of initial velocity experiments were analyzed by fitting data by nonlinear regression to the appropriate Equations 1-6 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 (30, 31), 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 4-6 apply to cooperative substrate saturation kinetics, where n is not affected by the presence of inhibitor (32).

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

(Eq. 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 for S; 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, and K_is2 are inhibition constants for the effect on S_0.5, where a suffix, 1 or 2, on K_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 min¹ mg¹.

Ligand Binding Studies-- Ligand binding was performed as described previously (33, 34). PRPP synthase (4.5 nmol) was incubated at pH 8.2 in 150 µl of 50 mM P_i, 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.

(Eq. 7)

(Eq. 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_B are 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stability of PRPP Synthase in the Absence of P_i-- PRPP synthase from E. coli is normally stored and diluted in the presence of 50 mM P_i to maintain stability (16). Therefore, to study P_i activation it was necessary to find a condition where the enzyme was stable in the absence of P_i. 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 P_i.

Influence of Mg²⁺ or Rib-5-P on P_i Activation-- Saturation with free Mg²⁺ resulted in nearly hyperbolic activation of PRPP synthase by P_i, whereas cooperative activation by P_i was observed at unsaturating free Mg²⁺ concentrations (Fig. 1A). Apparently, also the S_0.5 for P_i increased when the free Mg²⁺ concentration was lowered to 14 µM (Fig. 1A). Mg²⁺ activation appeared hyperbolic at P_i concentrations of 5 mM and above but was clearly cooperative at 1.5 mM P_i (Fig. 1B). However, the concentration for half-saturation with free Mg²⁺ changed by less than a factor of two over the entire range of P_i concentrations (Fig. 1B). The apparent cooperativity of P_i 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 P_i compared with the results obtained at 5 mM Rib-5-P (Fig. 2A). At 0.5 mM Rib-5-P, beginning inhibition by P_i 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 P_i concentration because increasing Rib-5-P concentrations induced partial substrate inhibition that was relieved by 50 mM P_i (Fig. 2B).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Activation of PRPP synthase by Pi or 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; Vapp = 130 ± 3, S0.5 = 3.1 ± 0.2 mM, n = 1.3 ± 0.1. , data were fitted to Equation 2; Vapp = 73 ± 1, S0.5 = 2.6 ± 0.1 mM, n = 2.0 ± 0.2. , data were fitted to Equation 2; Vapp = 39.1 ± 0.8, S0.5 = 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; Vapp = 129 ± 4, Km = 41 ± 4 µM. , data were fitted to Equation 1; Vapp = 103 ± 6, Km = 59 ± 10 µM. , data were fitted to Equation 2; Vapp = 55 ± 2, S0.5 = 69 ± 5 µM, n = 2.1 ± 0.2.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Saturation of PRPP synthase by Pi or 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; Vapp = 150 ± 6, S0.5 = 2.6 ± 0.3 mM, n = 1.2 ± 0.1. , data were fitted to Equation 2; Vapp = 129 ± 2, S0.5 = 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; Vapp = 157 ± 2, Km = 0.19 ± 0.01 mM. , , and , data were fitted to Equation 3.

Inhibition of P_i Activation by Ca²⁺, MgmATP, or cRib-5-P-- Divalent calcium, MgmATP, and cRib-5-P have been shown to competitively inhibit the binding of Mg²⁺, MgATP, and Rib-5-P, respectively (11). Results from experiments where P_i 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 P_i 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 P_i concentrations.

Inhibition of P_i Activation by ADP-- When P_i 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 on S_0.5 for P_i (Fig. 3 and Table I). The effect of ADP on both S_0.5 and V_app for saturation of the enzyme by P_i in the presence of 0.5 mM Rib-5-P could readily be determined when the data were fitted to Equation 5 (Fig. 3B and Table I). Data from ADP inhibition of P_i 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 P_i concentrations available to us (Fig. 3A and Table I). Increasing the concentration of P_i beyond 50 mM in the assay incubation results in the rapid formation of a MgP_i precipitate.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Inhibition of Pi activation of PRPP synthase by ADP. Assays were performed as described under "Experimental Procedures." Pi varied as indicated in the presence of the indicated concentrations of ADP and 5 mM Rib-5-P (A; data were fitted to Equation 5; the calculated constants are presented in Table I) or 0.5 mM Rib-5-P (B; data were fitted to Equation 6; the calculated constants are presented in Table I).

Binding of ADP-- An observed cooperativity in binding of MgmATP to PRPP synthase (14) 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 (11). To investigate whether the high degree of cooperativity associated with ADP binding to the allosteric site previously determined (14) 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 (14).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Binding of ADP to PRPP synthase. Binding experiments were performed as described under "Experimental Procedures." The binding of ADP was determined in the absence or presence of Rib-5-P as indicated. , data were fitted to Equation 7; Amax = 1.14 ± 0.05, KA = 59 ± 11 µM). , data were fitted to Equation 8; Amax = 1.0 ± 0.1, KA = 4±2 µM, Bmax = 1.0 ± 0.2, KB = 244 ± 52 µM, nb = 2.4 ± 1.4.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (8, 9, 11). Allosteric binding of ADP has been shown to occur either at conditions where the enzyme is fully saturated with Mg²⁺ and substrates (14-16) or prior to binding of Mg²⁺ and substrates as revealed by cooperative competitive ADP inhibition of Mg²⁺ activation (11).

By comparing MgmATP and ADP inhibition of P_i 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 P_i described by K_is1 and K_is2 is observed both at 0.5 and 5 mM Rib-5-P. This nonlinear effect of ADP on S_0.5 for P_i is sufficient to explain the inhibition by ADP at 5 mM Rib-5-P. Although a K_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 P_i, (Table I) suggests that ADP competes with P_i for binding to the regulatory site. However, because neither K_is1 nor K_is2 is comparable with K_is for MgmATP under similar conditions, we hesitate to further assign an exact physical meaning for K_is1 and K_is2. It appears that ADP and P_i 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 P_i 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 (30, 31, 35) with a preferred pathway in which P_i 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 P_i 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 P_i 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 P_i (i.e. before or after binding of P_i) 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 P_i. In favor of a fully random mechanism is the absence of inhibition at increasing P_i 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 P_i and if downstream binding of substrates and P_i also occurs to the enzyme inhibitor complex (36). Both Ca²⁺ (15) and MgmATP (11) 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 P_i binding therefore appears most consistent with the results presented here and those previously obtained as mentioned above.

In Scheme 1 an outline of the interaction of PRPP synthase with substrates, activators, and ADP is suggested. The allosteric effectors P_i 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 P_i is bound to the enzyme or not. The noncompetitive inhibition of P_i activation by Ca²⁺, MgmATP, and Rib-5-P is also consistent with the mechanism in Scheme 1.

View larger version (10K): [in this window] [in a new window]   Scheme 1.   Proposed steady state mechanism for a random binding of Pi to PRPP synthase comprising an ordered binding of Mg2+ and substrates. The bold line represents the fast pathway. E represents PRPP synthase. Pi (ADP) indicates that Pi and ADP compete for binding to the allosteric site.

According to Scheme 1 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. 1A where unsaturation of PRPP synthase with Mg²⁺ yields cooperative P_i activation can be explained by a preferred pathway in a random mechanism. However, unlike the saturation with Rib-5-P at low P_i concentrations (Fig. 2B), the Mg²⁺ activation at 1.5 mM P_i is cooperative (Fig. 1B), and no inhibition by increasing Mg²⁺ concentrations is observed. Because apparently no cooperativity is associated with Ca²⁺ inhibition of P_i activation, it may suggest that the apparent cooperativity of Mg²⁺ activation at low P_i is not due to homotropic site-site interactions. The apparent cooperativity of Mg²⁺ activation in the presence of 1.5 mM P_i (Fig. 1B) may be interpreted in terms of the apparent increase in S_0.5 for P_i at low Mg²⁺ (Fig. 1A). If the affinity of the enzyme for P_i 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 1 must somehow be a simplification of the actual overall mechanism for PRPP synthase. One consistent observation that is not explained by Scheme 1 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 mM P_i (11, 14). Our results from ADP inhibition of P_i activation at 0.5 mM and 5 mM Rib-5-P can be explained without including any cooperativity associated with the ADP inhibition. It seems controversial that ADP and P_i 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 1 other than those indicated, and because ADP and P_i apparently compete for binding to the same form(s) of the enzyme, this should be true for P_i as well. Apart from explaining the observed competition between binding of Mg²⁺ and ADP (11, 18) as a simple consequence of a random mechanism, Scheme 1 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 (17).

The recent solving of the crystal structure of B. subtilis PRPP synthase (25) 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 P_i, we may address specific questions about the regulatory mechanism in Scheme 1.

The mechanism in Scheme 1 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 P_i, it may suggest that the mechanism proposed in Scheme 1 can 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.

	ACKNOWLEDGEMENTS

We thank Jørgen Andresen for excellent technical assistance. K. Frank Jensen and Robert L. Switzer are acknowledged for valuable discussions.

	FOOTNOTES

^* This work was supported by the Danish National Research Foundation and the Danish Natural Science Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 45-35320239; Fax: 45-35320299; E-mail: martin@xray.ki.ku.dk.

Published, JBC Papers in Press, August 22, 2000, DOI 10.1074/jbc.M006346200

	ABBREVIATIONS

The abbreviations used are: PRPP, 5-phospho-D-ribosyl -1-diphosphate; Rib-5-P, ribose 5-phosphate; cRib-5-P, (+)-1-,2-,3--trihydroxy-4--cyclopentanemethanol 5-phosphate; mATP, ,-methylene ATP; MgmATP, ,-methylene MgATP.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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