©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Stable Carbocyclic Analog of 5-Phosphoribosyl-1-pyrophosphate to Probe the Mechanism of Catalysis and Regulation of Glutamine Phosphoribosylpyrophosphate Amidotransferase (*)

(Received for publication, March 7, 1995)

Jeong Hyun Kim (1), Dana Wolle (1)(§), Kochat Haridas (3)(¶), Ronald J. Parry (3), Janet L. Smith (2), Howard Zalkin (1)

From the  (1)Departments of Biochemistry and (2)Biological Sciences, Purdue University, West Lafayette, Indiana 47907 and the (3)Department of Chemistry, Rice University, Houston, Texas 77251

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-pyrophosphoryl-2-,3--dihydroxy-4--cyclopentanemethanol-5-phosphate (cPRPP). Although cPRPP competes with PRPP for binding to the catalytic C site of the Escherichia coli enzyme, two lines of evidence demonstrate that cPRPP, unlike PRPP, does not promote an active enzyme conformation. First, cPRPP was not able to ``activate'' Cys for reaction with glutamine or a glutamine affinity analog. The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys for catalysis. Second, binding of cPRPP to the C site blocks binding of AMP and GMP, nucleotide end product inhibitors, to this site. However, the binding of nucleotide to the allosteric site was essentially unaffected by cPRPP in the C site. Since it is expected that nucleotide inhibitors would bind with low affinity to the active enzyme conformation, the nucleotide binding data support the conclusion that cPRPP does not activate the enzyme.


INTRODUCTION

Glutamine phosphoribosylpyrophosphate (PRPP)()amidotransferase catalyzes the initial reaction in de novo purine nucleotide synthesis and is the key regulatory enzyme in the multistep pathway to AMP and GMP. Similar to other glutamine amidotransferases (Zalkin, 1993), NH can replace glutamine as amino donor in vitro (Messenger and Zalkin, 1979) and in vivo (Mäntsälä and Zalkin, 1984a; Mei and Zalkin, 1990). The reactions are glutamine + PRPP PRA + glutamate + PP and NH + PRPP PRA + PP.

The enzymes from Escherichia coli (Messenger and Zalkin, 1979) and Bacillus subtilis (Wong et al., 1981) have been purified to homogeneity and characterized (Zalkin, 1993). In addition, 13 glutamine PRPP amidotransferase sequences have been derived from cloned genes. These sequences are highly conserved (29-93% identity) leading to the conclusion that the enzymes in bacteria (Tso et al., 1982; Makaroff et al., 1983; Gu et al., 1992),()lower eukaryotes()(Mäntsälä and Zalkin, 1984b; Ludin et al., 1994), invertebrates (Clark, 1994), vertebrates (Brayton et al., 1994; Zhou et al., 1990; Iwahana et al., 1993) and plants (Ito et al., 1994; Kim et al., 1995) are homologous and are structurally similar. The glutamine PRPP amidotransferases from E. coli and B. subtilis are thus viewed as models for the enzyme from other organisms. The x-ray structure of the B. subtilis glutamine PRPP amidotransferase with bound nucleotide has been solved recently (Smith et al., 1994). The enzyme contains an NH-terminal glutamine-binding domain (N-domain) that is joined to a COOH-terminal domain (C-domain) with sites for NH-dependent synthesis of PRA and for end product inhibition by AMP and GMP. The N-domain has sequence and presumably structural similarity to the glutamine-binding N-domains of asparagine synthetase and glucosamine 6-phosphate synthase, enzymes that belong to a ``purF'' subfamily of amidotransferases (Zalkin, 1993). For this subfamily the N-domain contains an NH-terminal active site cysteine residue that is essential for transfer of the glutamine amide to the second substrate.

The C-domain contains two nucleotide sites, an allosteric A site in close proximity to a catalytic C site. The C site, which binds nucleotide in the crystal structure, is identified as a catalytic site by virtue of a PRPP-binding sequence motif (Hershey and Taylor, 1986; Hove-Jensen et al., 1986) which has also been identified as the active site in two other phosphoribosyltransferases (Scapin et al., 1994; Eads et al., 1994). Glutamine PRPP amidotransferase is not only a glutamine amidotransferase but is also a phosphoribosyltransferase. PRPP has been modeled into the glutamine PRPP amidotransferase C site in a position occupied by the ribose-5-phosphate moiety of AMP (Smith et al., 1994). The proximal A site, identified by bound nucleotide in the crystal structure, is between subunits of the enzyme tetramer. The proximity of the A and C sites is unusual and may contribute to the synergistic binding of AMP and GMP that is responsible for the synergistic end product inhibition (Zhou et al., 1994).

The mechanism of amide transfer from glutamine is unknown and it is an open question whether glutamine hydrolysis precedes, is concerted with, or occurs after attachment of the NH group to C-1 of PRPP. In the crystal structure of the inhibited B. subtilis glutamine PRPP amidotransferase containing bound AMP, the catalytic thiol of Cys in the N-domain and C-1 of PRPP modeled into the C-domain are separated by a 16-Å solvent-filled space (Fig. 1). There are thus at least two barriers to catalysis. First, the inhibited enzyme is in a conformation described as ``open'' in which the glutamine and PRPP sites are too far apart for catalysis. Second, although Cys is proximal to the interdomain boundary, its sulfhydryl group is sequestered in a cavity within the N-domain where it is inaccessible to glutamine. Given the very low rates of glutamine hydrolysis and reaction with glutamine affinity analogs in the absence of PRPP (Messenger and Zalkin, 1979), the latter substrate must somehow ``activate'' Cys for the initial steps of glutamine amide transfer.


Figure 1: A, ribbon diagram of the glutamine PRPP amidotransferase subunit in the open conformation. The N-domain with the catalytic residue Cys is shown at the top of the diagram and the C-domain with PRPP modeled into the C-site at the bottom. The catalytic thiol of Cys and C-1 of PRPP are 16-Å apart. strands are depicted as arrows, -helices as spirals, and other structures as a tube. Side chain atoms are shown for Cys, Arg, and Tyr. A 30-residue loop, which would obscure the PRPP site but does not contact it, is omitted for clarity. B, close-up view of the active site. The view is the same as in A with additional side chains from the PRPP-binding sequence fingerprint (Asp, Asp, Ser, Ile, Val, Arg, and Gly) included. Atom coloring in both A and B: C, black; O, red; N, blue; S, yellow; and P, green. Diagrams were made with the program Molscript (Kraulis, 1991).



Due to chemical lability it has not been possible to investigate how binding of PRPP to the glutamine PRPP amidotransferase C site activates Cys for catalysis and what effect this interaction has on binding of nucleotides to the A and C sites. The present approach was to utilize a new stable PRPP analog, cPRPP, to further examine the geometry of the C site, the requirements for activation of Cys for amide transfer and the effect that a substrate analog has on nucleotide binding. The results demonstrate that cPRPP competes with PRPP for the C site and in addition can block binding of nucleotide to the C site. Yet, unlike PRPP, cPRPP does not activate Cys for catalysis. Furthermore, a mutation that reduces the affinity of nucleotide for the C site has no appreciable affect on PRPP or cPRPP binding, thus supporting the assignment of distinct ribose phosphate and purine base binding regions in the C site (Smith et al., 1994).


EXPERIMENTAL PROCEDURES

Synthesis of cPRPP

The PRPP analog cPRPP was synthesized as reported (Parry and Haridas, 1993). In cPRPP a methylene carbon replaces the ring oxygen of PRPP. The compound was racemic because the starting materials employed in the synthesis were not asymmetric, and no resolutions were carried out. The compound used in these studies therefore consists of a mixture of equal parts of the two possible enantiomers, one of which corresponds in absolute configuration to the configuration of natural PRPP.

Enzyme Purification

Wild type E. coli glutamine PRPP amidotransferase and the P410W C site mutant enzyme were overproduced from plasmid pGZ14 and its mutant derivative, respectively, in E. coli strain TX358 (purF recA) as described (Zhou et al., 1993). Cells were harvested in late log phase from cultures grown in 2-liter flasks and were stored at -20 °C prior to purification. The enzyme was purified (Mei and Zalkin, 1989) to approximately 95% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970). Enzyme at a concentration of approximately 20 mg/ml was stored at -20 °C. Protein concentration was determined by A using the value 8.12 for a 1% solution (Messenger and Zalkin, 1979). Specific activity was typically 15-20 units/mg protein for the wild type enzyme and 7.5 units/mg protein for the P410W enzyme. A unit of activity is defined as the amount of enzyme needed to produce 1.0 µmol of glutamate/min.

Enzyme Assay

Glutamine-dependent activity was determined by measurement of the glutamate produced (Messenger and Zalkin, 1979). Reactions contained 0-0.5 mM PRPP (Sigma), 10 mM glutamine, 5 mM MgCl, 0-1.0 mM cPRPP, 50 mM Tris-HCl (pH 8.0) and approximately 40 ng of enzyme in a total volume of 0.1 ml. Incubation was at 37 °C, usually for 10 min. Reactions were quenched in a boiling water bath for 30 s, and glutamate was determined by the glutamate dehydrogenase method (Messenger and Zalkin, 1979). Glutaminase activity in the absence of PRPP was determined by a similar procedure, but the enzyme concentration was increased 10-100-fold due to the low rate of reaction and samples were removed at timed intervals to accurately determine the rate of glutamate production. All assays were shown to be linear for at least 12 min, and rates were proportional to enzyme concentration over at least a 4-fold range.

Inactivation by DON

Enzyme (approximately 8 µg) was incubated at room temperature for 0-30 min in a mixture containing 1 mg/ml bovine serum albumin, 50 mM potassium phosphate (pH 7.5), 10 µM DON (Sigma) if present, 1.0 mM PRPP if present, and 1.0 mM cPRPP, if present, in a total volume of 0.1 ml. At specific times, 5-µl aliquots were removed and enzyme activity remaining was determined in the standard 0.5-ml assay for glutamate production (Messenger and Zalkin, 1979). The 100-fold dilution quenched the reaction of enzyme with DON.

Nucleotide Binding

Nucleotide binding was determined by equilibrium dialysis (Zhou et al., 1994) using chambers of 100 µl. Chambers were separated by a 12,000-14,000 molecular weight cut off dialysis membrane (Spectrapore) that was stored in 200 mM Tris-HCl (pH 7.5), 20 mM MgCl, and 0.01% sodium azide at 4 °C. The membrane was blotted on filter paper before loading between the chambers. One chamber contained 200 mM Tris-HCl (pH 7.5), 20 mM MgCl, 28 nM [2,8-H] AMP, or 35 nM [8-H]GMP (0.1 µCi), 0-4.6 mM unlabeled AMP or GMP, and 1.3 or 2.0 mM cPRPP, if present, in a total volume of 50 µl. The other chamber contained 10 mM Tris-HCl (pH 7.5) and 200-300 µM enzyme subunit in a total volume of 50 µl. Radioactive purine nucleotides were purchased from Moravek Biochemicals, Inc. Dialysis was for 20 h at 4 °C in a rotating apparatus. Samples of 40 µl were removed from each chamber and were counted for radioactivity.

Data Analysis

Data for substrate saturation by PRPP were fit to the Michaelis-Menten equation using Ultrafit software (Biosoft, Cambridge, United Kingdom). K was also calculated by the graphical method of Dixon (Dixon and Webb, 1979) using Ultrafit. Equilibrium binding data were fitted to the Scatchard equation (Scatchard, 1949) by non-linear regression using Ultrafit.


RESULTS

Inhibition by cPRPP

cPRPP was evaluated as a PRPP analog for glutamine PRPP amidotransferase. The data in Fig. 2show that inhibition of the wild type enzyme by cPRPP was competitive with PRPP. The K for PRPP of 53 ± 12 µM agrees with the value of 67 µM determined previously (Messenger and Zalkin, 1979). Inhibition data analyzed graphically by the method of Dixon (Dixon and Webb, 1979), shown in Fig. 3, yield a K of 116 ± 25 µM for cPRPP. Since the cPRPP preparation is a 1:1 racemic mixture (Parry and Haridas, 1993), the actual K for cPRPP is 58 ± 13 µM, a value that is similar to the K for PRPP. We also examined the inhibition of a C site mutant enzyme by cPRPP. The P410W replacement in the C site decreases the binding affinity of AMP for the C site by 5-fold with a corresponding decrease in enzyme inhibition by AMP (Zhou et al., 1994). Inhibition of P410W by cPRPP was competitive with PRPP (data not shown). The K of PRPP was 31 ± 5 µM and the K for cPRPP was 53 ± 5 µM, values not significantly different than those obtained for the wild type. Thus, cPRPP competes effectively with PRPP for the amidotransferase C site.


Figure 2: Inhibition by cPRPP. Enzyme activity was determined as described under ``Experimental Procedures.'' Symbols are , no added cPRPP; , 0.1 mM cPRPP added. Data were plotted using the Lineweaver-Burke equation. V is in units/mg protein.




Figure 3: Calculation of the K for cPRPP. Enzyme activity was assayed with either 50 µM () or 100 µM () PRPP and varied cPRPP. Data were plotted using the graphical method of Dixon(17) . V is in units/mg protein.



Glutamine Hydrolysis

Synthesis of phosphoribosylamine is tightly coupled to glutamine hydrolysis. In contrast, some amidotransferases catalyze a glutaminase activity in which glutamine hydrolysis can be uncoupled from product formation (Zalkin, 1993). For example, glutamine hydrolysis in anthranilate synthase, a ``trpG'' subfamily amidotransferase, is dependent upon chorismate, the second substrate, and is assayed in the presence of EDTA which blocks synthesis of anthranilate (Nagano et al., 1970). In cases where the rate of glutaminase activity is comparable to the overall rate of product formation, such as in the reaction catalyzed by anthranilate synthase, glutamine hydrolysis may reflect a step in the overall reaction. An active site cysteine is required for reaction of glutamine. For glutamine PRPP amidotransferase, the rate of PRPP-independent glutaminase activity was reported to be about 4% of the overall rate (Messenger and Zalkin, 1979). The limited capacity for glutamine hydrolysis in the absence of PRPP can now be explained by the finding of an inactive conformation for the nucleotide-inhibited enzyme (no PRPP in the C site) in which Cys is sequestered and is inaccessible to glutamine (Smith et al., 1994). This information strongly implies that binding of PRPP to the C site is needed to ``activate'' Cys. The effect of PRPP on glutaminase activity could not be determined previously since there was no way to uncouple glutamine hydrolysis from phosphoribosylamine production. We have therefore reinvestigated the glutaminase activity to determine whether cPRPP bound to the PRPP site can activate Cys for glutamine hydrolysis. Data summarized in Table 1show a basal rate of PRPP-independent glutaminase that was 0.8% of the overall rate for reaction of glutamine with PRPP. This low rate of glutaminase was not stimulated by cPRPP. Ribose 5-P, on the other hand, stimulated the rate of glutaminase by 10-fold, as had been observed earlier (Messenger and Zalkin, 1979).



Affinity Labeling of Cys

Cys is an active site residue required for glutamine amide transfer. Affinity labeling of Cys with the glutamine analog DON is presumed to mimic the nucleophilic attack of Cys on the carboxamide of glutamine, resulting in release of ammonia and formation of a hypothetical -glutamyl-enzyme covalent intermediate (Nagano et al., 1970; Zalkin, 1993). Nucleophilic attack of Cys on the -carbon of DON results in displacement of molecular dinitrogen and alkylation of Cys. The data in Fig. 4show, as reported previously (Messenger and Zalkin, 1979), that affinity labeling of Cys by DON was dependent upon PRPP. cPRPP did not support the reaction of DON with Cys, confirming that cPRPP did not activate Cys.


Figure 4: Alkylation of Cys by DON. Enzyme was incubated with DON, as described under ``Experimental Procedures.'' Reactions with PRPP are shown by (), those with no PRPP, with cPRPP, or a control that did not contain DON, are all shown by (). Samples were removed from each incubation and were assayed for activity.



Effect of cPRPP on Nucleotide Binding

Glutamine PRPP amidotransferase is subject to allosteric end product inhibition by adenine and guanine nucleotides. AMP and GMP can each bind to two sites/subunit, the allosteric A site and catalytic C site (Zhou et al., 1994). Synergistic inhibition by AMP plus GMP results from synergistic binding of AMP to the C site and GMP to the A site. Availability of the stable competitive inhibitor cPRPP affords the opportunity to examine directly the expected competition between substrate analog and nucleotide for the C site and the consequences that cPRPP binding to the C site has on nucleotide binding to the A site. The lability of PRPP has precluded its use in equilibrium binding experiments. Equilibrium binding of GMP to the enzyme is shown in Fig. 5. GMP binding extrapolated to 1.7 eq nucleotide/subunit with a K of 220 µM (Table 2). In contrast to earlier experiments (Zhou et al., 1994), the K values of GMP for the A and C sites were not sufficiently different to be resolved. We infer that GMP was bound equally to the A and C sites (Table 2). In the presence of cPRPP the binding stoichiometry was reduced to 0.90 eq GMP/subunit. It is therefore reasonable to infer that binding of cPRPP to the C site blocked nucleotide binding to this site and marginally decreased the affinity of GMP for the A site. In a similar manner, cPRPP blocked binding of AMP to the C site (Table 2). In this case, however, perhaps as a result of an increased K, binding of AMP to the A site was not detected. Under the experimental conditions employed, binding of a ligand with a K > 1.0 mM would not be detected.


Figure 5: Effect of cPRPP on GMP binding. Equilibrium dialysis was performed with 1.0 mM cPRPP () or without cPRPP (). Enzyme concentration was 84.25 µM (337 µM subunit).





To reduce the potential ambiguity of assigning bound nucleotide to A and C sites, we examined the effect of cPRPP on nucleotide binding to the P410W mutant. This C site replacement effectively decreases, below the limit of detection, the capacity of a single nucleotide to bind to the C site (Zhou et al., 1994). However, approximately 1 eq of GMP could bind to the A site of this mutant and thus permitted synergistic binding of AMP to the mutant C site with a 5-fold increased K compared to the wild type. This suggests that the binding affinity for nucleotide to the mutant C site is reduced about 5-fold. Data in Table 2, line 5, show binding of 0.83 eq of GMP to the P410W mutant enzyme. The bound GMP is assigned to the A site by virtue of the preference of GMP for the A site (Zhou et al., 1994) and the disabling mutation in the C site. Binding of GMP to the P410W A site was unperturbed by cPRPP (Table 2, line 6). Given the competitive inhibition and unchanged K for cPRPP, it is safe to infer saturation of the P410W C site by cPRPP. We next determined the capacity of GMP to bind to the A site under conditions that mimic saturation of the enzyme with the two substrates. For this purpose Cys was alkylated with the glutamine affinity analog DON until less than 3.2% of the glutamine-dependent activity remained. The enzyme was dialyzed to remove PRPP and residual unbound DON, and GMP binding was determined in the presence or absence of cPRPP. Data in Table 2, lines 7 and 8, show that the two substrate analogs had no significant effect on binding of GMP to the A site. The implications of this result on the capacity of these substrate analogs to promote an active enzyme conformation and influence nucleotide binding are discussed below.


DISCUSSION

cPRPP is a stable substrate analog that competes with PRPP for the glutamine PRPP amidotransferase catalytic site. Interactions important for binding of PRPP to the C site can be inferred from the structural model of the homologous B. subtilis amidotransferase (Smith et al., 1994). All amino acids that interact with PRPP modeled into the C site are conserved in the E. coli enzyme and in all other glutamine PRPP amidotransferases for which sequences are available. These include interactions of Lys with the pyrophosphate -phosphate, Arg with the 5-phosphate, and the 2- and 3-hydroxyls of PRPP with a Mg ion. In addition, Thr, Asp, and Asp, all numbered according to the E. coli sequence, and a water molecule contribute to the octahedral ligand field of the Mg. PRPP has been modeled into the C site with a C3-endo pucker to conform with the conformation of the ribose phosphate moiety of bound AMP (Smith et al., 1994). Although it is not known whether the conformations of cPRPP and PRPP are identical, it is reasonable to expect the same groups to be involved in binding the substrate and substrate analog to the glutamine PRPP amidotransferase C site.

The results of experiments reported here support the view that cPRPP competes with PRPP for the C site. First, enzyme inhibition was competitive with PRPP in the wild type and in a mutant with a partially disabled C site having a higher K for nucleotides. The K for cPRPP was not significantly different in the wild type and mutant enzymes, and these values were similar to the K for PRPP in each case. Second, binding of cPRPP to the C site excluded binding of nucleotides to this site. Given these results we determined whether binding of cPRPP to the C site could promote formation of an active ``closed'' conformation of the enzyme. Two features are expected to distinguish closed and open conformations. (i) The ability of Cys to participate in glutamine hydrolysis and amide transfer will differ. In the feedback-inhibited inactive open conformation seen in the crystal structure, Cys is sequestered (Smith et al., 1994) and is inaccessible to glutamine or DON, a glutamine affinity analog. Additionally, in this open conformation, the positions of Cys and bound PRPP are too far apart for N-transfer (Smith et al., 1994). Our working hypothesis is that PRPP is required both to activate Cys for reaction with glutamine or DON by making it sterically accessible and to more favorably position the glutamine and PRPP sites for amide transfer. (ii) Nucleotide binding to closed and open enzyme conformations should differ. Based on the inaccessibility of Cys and its separation from the PRPP-binding site, it appears unlikely that substrates can bind with high affinity to the inhibited enzyme having nucleotides in the A and C sites (Smith et al., 1994). Nucleotide bound to the allosteric A site which is between subunits of the tetrameric enzyme is likely responsible for the open conformation of the active site seen in the crystal structure of the inhibited enzyme. Likewise, nucleotides are expected to have a low binding affinity for the enzyme-substrate complex in the closed active conformation.

By both criteria, cPRPP did not promote formation of the active conformation. First, binding of cPRPP to the C site did not activate Cys for reaction with glutamine or DON. Second, although binding of cPRPP to the C site excluded binding of AMP to this site in the wild type enzyme, binding of AMP or GMP to the A site was not perturbed in the wild type or in the P410W C site mutant, suggesting that the cPRPPenzyme complex was not in the active conformation. These results have implications for understanding catalysis and inhibition by nucleotides and are discussed below.

There are at least three possibilities to account for the failure of cPRPP to activate Cys for reaction with glutamine and DON. First, cPRPP may assume a conformation different from that of PRPP, and consequently binding to the C site may not be identical for the substrate and substrate analog. This could result in different ligand-protein interactions that might account for the different capacities to activate. The competitive kinetics for cPRPP inhibition and the similarity of K for PRPP and K for cPRPP argue against, but do not eliminate, this possibility. Second, a structural difference between PRPP and cPRPP could prevent the conformational transition between open and closed states of the enzyme. cPRPP does not contain the furanose ring oxygen atom that is present in PRPP. A specific interaction of the furanose ring oxygen of PRPP with the protein may contribute to the conformational change needed to activate Cys. This would explain the limited capacity of ribose 5-phosphate but not cPRPP to activate Cys. Whether altered ligand-protein interactions result from different cPRPP conformations or different functional groups cannot be distinguished presently and must await crystallographic analysis.

The best candidate for a hydrogen-bond donor to the ring oxygen of PRPP is the side chain of Tyr, a conserved residue in all glutamine PRPP amidotransferase sequences. In the crystal structure Tyr is positioned close to the furanose ring oxygen of the ribose moiety of AMP in the C site or of PRPP modeled into this site (Fig. 1). Tyr is connected directly to the cavity in which Cys is sequestered via Arg, a residue that lines one wall of the cavity and is also invariant. A hydrogen bond between the Tyr hydroxyl and the ring oxygen of PRPP may facilitate a structural change that unmasks Cys for reaction with glutamine. Affinity labeling and mutagenesis experiments support a role for Tyr in glutamine amide transfer. Tyr was one of the residues affinity labeled by 5`-p-fluorosulfonylbenzoyladenosine (Zhou et al., 1993). Labeling by 5`-p-fluorosulfonylbenzoyladenosine bound to a nucleotide site places Tyr in proximity to the C site. Replacement of Tyr by Ala resulted in an enzyme that retained 75% of the wild type activity using NH as substrate but retained only 1% activity with glutamine. Thus Tyr is needed not for binding of PRPP to the C site, but rather for glutamine amide transfer. An interaction of Tyr with the ring oxygen of PRPP may contribute to activation of Cys.

A third possibility to explain the inability of cPRPP to activate Cys is that the reactivity at C1 of PRPP is important for the activation of Cys and amide transfer from glutamine. If formation of a hypothetical Cys-glutamine tetrahedral intermediate were in some way coupled to the availability of a site to accept the amide, cPRPP would not serve this function and reaction of Cys with glutamine or DON would not occur. cPRPP, unlike PRPP, is not subject to nucleophilic attack at carbon 1 with displacement of pyrophosphate.

The fact that binding of cPRPP to the C site did not significantly affect nucleotide binding to the A site has implications for feedback regulation. We favor the explanation that the small differences in K for nucleotide binding to the A site in the presence and absence of cPRPP indicate that binding of cPRPP did not result in the closed enzyme conformation having low affinity for nucleotide binding. We consider unlikely the possibility that nucleotides bind with similar affinity to the A site in active closed and inactive open conformations. In this light there are three conclusions to be derived from the data in Table 2. First, alkylation of Cys with DON which mimics the inferred enzyme-glutamate covalent intermediate in catalysis (Zalkin, 1993) did not assist formation of the closed conformation having low affinity nucleotide binding to the A site, in the presence or absence of cPRPP. Thus, binding of PRPP, not glutamine, appears to be the key step for enzyme activation. Second, the K for binding of GMP to the A site was similar in the wild type, with or without cPRPP in the C site, and in the P410W enzyme with or without cPRPP in the C site. Thus, GMP binding to the A site is not dependent on nucleotide in the C site. Finally, the P410W mutation is seen to reduce the affinity of nucleotide but not cPRPP for the C site. This is consistent with the x-ray structural model of the C site in which PRPP binds to the position occupied by the ribose phosphate moiety of the nucleotide (Smith et al., 1994).

The present data for nucleotide binding in the presence of cPRPP support earlier conclusions on the binding selectivity of the A and C sites obtained with mutants (Zhou et al., 1994). With cPRPP bound to the C site, GMP exhibited a higher affinity than AMP for the A site. Thus, although GMP and AMP can each bind to both sites, there is a preference of GMP for the A site and of AMP for the C site. This selectivity combined with the effect that binding of one nucleotide has on the binding of the other accounts for the synergistic feedback inhibition of the enzyme by AMP plus GMP. Although the competition between cPRPP and nucleotide for the C site did not prevent access of GMP for the A site, binding affinity is reduced significantly in the absence of synergism.


FOOTNOTES

*
This research was supported by United States Public Health Service Grants GM24658 (to H. Z.), DK42303 (to J. L. S.), and GM26569 and a grant from the Robert A. Welch Foundation (to R. J. P.). This is Journal Paper 14650 from the Purdue University Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Blood Research Institute, 8727 Watertown Plank Rd., Wauwatosa, WI 53226.

Present address: Bionumerik Pharmaceuticals, Inc., 8122 Datapoint, #1250, San Antonio, TX 78229.

The abbreviations used are: PRPP, 5-phosphoribosyl-1-pyrophosphate; PRA, 5-phosphoribosyl-1--amine; cPRPP, 1--pyrophosphoryl-2-, 3--dihydroxy-4--cyclopentanemethanol-5-phosphate; DON, 6-diazo-5-oxo-L-norleucine.

Lactobacillus PurF sequence corrected in GenBank M85265[GenBank® Link].

Neurospora crassa; Dan Ebbole, personal communication.


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