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• W2094647907 abstract "Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-αpyrophosphoryl-2-α,3-α-dihydroxy-4-β-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” Cys1 for reaction with glutamine or a glutamine affinity analog. The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys1 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. Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-αpyrophosphoryl-2-α,3-α-dihydroxy-4-β-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” Cys1 for reaction with glutamine or a glutamine affinity analog. The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys1 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. Glutamine phosphoribosylpyrophosphate (PRPP)1 1The abbreviations used are: PRPP5-phosphoribosyl-1-pyrophosphatePRA5-phosphoribosyl-1-β-aminecPRPP1-α-pyrophosphoryl-2-α, 3-α-dihydroxy-4-β-cyclopentanemethanol-5-phosphateDON6-diazo-5-oxo-L-norleucine. 1The abbreviations used are: PRPP5-phosphoribosyl-1-pyrophosphatePRA5-phosphoribosyl-1-β-aminecPRPP1-α-pyrophosphoryl-2-α, 3-α-dihydroxy-4-β-cyclopentanemethanol-5-phosphateDON6-diazo-5-oxo-L-norleucine. 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), NH3 can replace glutamine as amino donor in vitro (Messenger and 29Zalkin H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 66: 203-309PubMed Google Scholar) and in vivo (Mäntsälä and 15Mäntsälä P. Zalkin H. J. Biol. Chem. 1984; 259: 14230-14236Abstract Full Text PDF PubMed Google Scholar; 18Mei B. Zalkin H. J. Bacteriol. 1990; 172: 3512-3514Crossref PubMed Google Scholar). The reactions are glutamine + PRPP → PRA + glutamate + PPi and NH3+ PRPP → PRA + PPi.The enzymes from Escherichia coli (19Messenger L.J. Zalkin H. J. Biol. Chem. 1979; 254: 3382-3392Abstract Full Text PDF PubMed Google Scholar) and Bacillus subtilis (28Wong J.Y. Bernlohr D.A. Turnbough C.L. Switzer R.L. Biochemistry. 1981; 20: 5669-5674Crossref PubMed Scopus (14) Google Scholar) 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 (27Tso J.Y. Zalkin H. vanCleemput M. Yanofsky C. Smith J.M. J. Biol. Chem. 1982; 257: 3525-3531Abstract Full Text PDF PubMed Google Scholar; 14Makaroff C.A. Zalkin H. Switzer R.L. Vollmer S.J. J. Biol. Chem. 1983; 258: 10586-10593Abstract Full Text PDF PubMed Google Scholar; 5Gu Z.-M.M. Martindale D.W. Lee B.H. Gene (Amst.). 1992; 119: 123-126Crossref PubMed Scopus (11) Google Scholar),2 2Lactobacillus PurF sequence corrected in GenBank M85265[GenBank® Link]. 2Lactobacillus PurF sequence corrected in GenBank M85265[GenBank® Link]. lower eukaryotes3 3Neurospora crassa; Dan Ebbole, personal communication. 3Neurospora crassa; Dan Ebbole, personal communication.(Mäntsälä and 16Mäntsälä P. Zalkin H. J. Biol. Chem. 1984; 259: 8478-8484Abstract Full Text PDF PubMed Google Scholar; 13Ludin K.M. Hilti N. Schweingruber M.E. Curr. Genet. 1994; 25: 465-468Crossref PubMed Scopus (4) Google Scholar), invertebrates (2Clark D.V. Genetics. 1994; 136: 547-557Crossref PubMed Google Scholar), vertebrates (1Brayton K.A. Chen Z. Zhou G. Nagy P. Gavalas A. Trent J.M. Deaven L.L. Dixon J.E. Zalkin H. J. Biol. Chem. 1994; 269: 5313-5321Abstract Full Text PDF PubMed Google Scholar; 30Zhou G. Dixon J.E. Zalkin H. J. Biol. Chem. 1990; 265: 21152-21159Abstract Full Text PDF PubMed Google Scholar; 9Iwahana H. Yamaoka T. Mizutani M. Mizusawa N. Ii S. Yoshimoto K. Itakura M. J. Biol. Chem. 1993; 268: 7225-7237Abstract Full Text PDF PubMed Google Scholar) and plants (8Ito T. Shiraishi H. Okada K. Shimura Y. Plant Mol. Biol. 1994; 26: 529-533Crossref PubMed Scopus (21) Google Scholar; 10Kim J.H. Delauney A.J. Verma D.P.S. Plant J. 1995; 7: 77-86Crossref PubMed Scopus (22) Google Scholar) 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 (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar). The enzyme contains an NH2-terminal glutamine-binding domain (N-domain) that is joined to a COOH-terminal domain (C-domain) with sites for NH3-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 NH2-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 (6Hershey H.V. Taylor M.W. Gene (Amst.). 1986; 43: 287-293Crossref PubMed Scopus (108) Google Scholar; 7Hove-Jensen B. Harlow K.W. King C.J. Switzer R.L. J. Biol. Chem. 1986; 261: 6765-6771Abstract Full Text PDF PubMed Google Scholar) which has also been identified as the active site in two other phosphoribosyltransferases (23Scapin G. Grubmeyer C. Sacchettini J.C. Biochemistry. 1994; 33: 1287-1294Crossref PubMed Scopus (114) Google Scholar; 4Eads J.C. Scapin G. Xu Y. Grubmeyer C. Sacchettini J.C. Cell. 1994; 78: 325-334Abstract Full Text PDF PubMed Scopus (205) Google Scholar). 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 (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar). 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 (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar).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 NH2 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 Cys1 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 Cys1 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” Cys1 for the initial steps of glutamine amide transfer.Due to chemical lability it has not been possible to investigate how binding of PRPP to the glutamine PRPP amidotransferase C site activates Cys1 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 Cys1 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 Cys1 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 (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar).EXPERIMENTAL PROCEDURESSynthesis of cPRPPThe PRPP analog cPRPP was synthesized as reported (21Parry R.J. Haridas K. Tetrahedron Lett. 1993; 34: 7013-7016Crossref Scopus (10) Google Scholar). 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 PurificationWild 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 (31Zhou G. Charbonneau H. Colman R.F. Zalkin H. J. Biol. Chem. 1993; 268: 10471-10481Abstract Full Text PDF PubMed Google Scholar). 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 (12Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar). Enzyme at a concentration of approximately 20 mg/ml was stored at −20°C. Protein concentration was determined by A278 using the value 8.12 for a 1% solution (Messenger and 19Messenger L.J. Zalkin H. J. Biol. Chem. 1979; 254: 3382-3392Abstract Full Text PDF PubMed Google Scholar). 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 AssayGlutamine-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 MgCl2, 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 DONEnzyme (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 BindingNucleotide 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 MgCl2, 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 MgCl2, 28 nM [2,8-3H] AMP, or 35 nM [8-3H]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 AnalysisData for substrate saturation by PRPP were fit to the Michaelis-Menten equation using Ultrafit software (Biosoft, Cambridge, United Kingdom). Ki was also calculated by the graphical method of Dixon (3Dixon M. Webb E.C. Enzymes. Longman Group, Ltd., London1979: 350-352Google Scholar) using Ultrafit. Equilibrium binding data were fitted to the Scatchard equation (24Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17759) Google Scholar) by non-linear regression using Ultrafit.RESULTSInhibition by cPRPPcPRPP was evaluated as a PRPP analog for glutamine PRPP amidotransferase. The data in Fig. 2 show that inhibition of the wild type enzyme by cPRPP was competitive with PRPP. The Km 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 Ki of 116 ± 25 μM for cPRPP. Since the cPRPP preparation is a 1:1 racemic mixture (Parry and Haridas, 1993), the actual Ki for cPRPP is 58 ± 13 μM, a value that is similar to the Km 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 Km of PRPP was 31 ± 5 μM and the Ki 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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(17Mei B. Zalkin H. J. Biol. Chem. 1989; 264: 16613-16619Abstract Full Text PDF PubMed Google Scholar). V is in units/mg protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Glutamine HydrolysisSynthesis 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 (20Nagano H. Zalkin H. Henderson E.J. J. Biol. Chem. 1970; 245: 3810-3820Abstract Full Text PDF PubMed Google Scholar). 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 Cys1 is sequestered and is inaccessible to glutamine (26Smith J.L. Zaluzec E.J. Wery J-P. Niu L. Switzer R.L. Zalkin H. Satow Y. Science. 1994; 264: 1427-1433Crossref PubMed Scopus (226) Google Scholar). This information strongly implies that binding of PRPP to the C site is needed to “activate” Cys1. 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 Cys1 for glutamine hydrolysis. Data summarized in Table 1 show 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).TABLE I Open table in a new tab Affinity Labeling of Cys1Cys1 is an active site residue required for glutamine amide transfer. Affinity labeling of Cys1 with the glutamine analog DON is presumed to mimic the nucleophilic attack of Cys1 on the carboxamide of glutamine, resulting in release of ammonia and formation of a hypothetical γ-glutamyl-enzyme covalent intermediate (20Nagano H. Zalkin H. Henderson E.J. J. Biol. Chem. 1970; 245: 3810-3820Abstract Full Text PDF PubMed Google Scholar; 29Zalkin H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 66: 203-309PubMed Google Scholar). Nucleophilic attack of Cys1 on the δ-carbon of DON results in displacement of molecular dinitrogen and alkylation of Cys1. The data in Fig. 4 show, as reported previously (Messenger and Zalkin, 1979), that affinity labeling of Cys1 by DON was dependent upon PRPP. cPRPP did not support the reaction of DON with Cys1, confirming that cPRPP did not activate Cys1.Figure 4:Alkylation of Cys1 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of cPRPP on Nucleotide BindingGlutamine 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 (30Zhou G. Dixon J.E. Zalkin H. J. Biol. Chem. 1990; 265: 21152-21159Abstract Full Text PDF PubMed Google Scholar). 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 Kd of 220 μM (Table 2). In contrast to earlier experiments (1Brayton K.A. Chen Z. Zhou G. Nagy P. Gavalas A. Trent J.M. Deaven L.L. Dixon J.E. Zalkin H. J. Biol. Chem. 1994; 269: 5313-5321Abstract Full Text PDF PubMed Google Scholar), the Kd 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 Kd, binding of AMP to the A site was not detected. Under the experimental conditions employed, binding of a ligand with a Kd > 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE II Open table in a new tab 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 (1Brayton K.A. Chen Z. Zhou G. Nagy P. Gavalas A. Trent J.M. Deaven L.L. Dixon J.E. Zalkin H. J. Biol. Chem. 1994; 269: 5313-5321Abstract Full Text PDF PubMed Google Scholar). 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 Kd 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 (1Brayton K.A. Chen Z. Zhou G. Nagy P. Gavalas A. Trent J.M. Deaven L.L. Dixon J.E. Zalkin H. J. Biol. Chem. 1994; 269: 5313-5321Abstract Full Text PDF PubMed Google Scholar) 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 Ki 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 Cys1 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.DISCUSSIONcPRPP 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 (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar). 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 Lys349 with the pyrophosphate β-phosphate, Arg371 with the 5-phosphate, and the 2- and 3-hydroxyls of PRPP with a Mg2+ ion. In addition, Thr304, Asp366, and Asp367, all numbered according to the E. coli sequence, and a water molecule contribute to the octahedral ligand field of the Mg2+. 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 (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar). 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 Kd for nucleotides. The Ki for cPRPP was not significantly different in the wild type and mutant enzymes, and these values were similar to the Km 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 Cys1 to participate in glutamine hydrolysis and amide transfer will differ. In the feedback-inhibited inactive open conformation seen in the crystal structure, Cys1 is sequestered (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar) and is inaccessible to glutamine or DON, a glutamine affinity analog. Additionally, in this open conformation, the positions of Cys1 and bound PRPP are too far apart for N-transfer (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar). Our working hypothesis is that PRPP is required both to activate Cys1 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 Cys1 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 (32Zhou G. Smith J.L. Zalkin H. J. Biol. Chem. 1994; 269: 6784-6789Abstract Full Text PDF PubMed Google Scholar). 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 Cys1 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 cPRPP♦enzyme 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 Cys1 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 t" @default.
• W2094647907 created "2016-06-24" @default.
• W2094647907 creator A5015889970 @default.
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• W2094647907 date "1995-07-01" @default.
• W2094647907 modified "2023-09-29" @default.
• W2094647907 title "A Stable Carbocyclic Analog of 5-Phosphoribosyl-1-pyrophosphate to Probe the Mechanism of Catalysis and Regulation of Glutamine Phosphoribosylpyrophosphate Amidotransferase" @default.
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