©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inactivation of Escherichia coli Phosphoribosylpyrophosphate Synthetase by the 2`,3`-Dialdehyde Derivative of ATP
IDENTIFICATION OF ACTIVE SITE LYSINES (*)

(Received for publication, May 23, 1995; and in revised form, June 30, 1995)

Ida Hilden Bjarne Hove-Jensen Kenneth W. Harlow (§)

From the Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, DK-1353 Copenhagen K, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The enzyme 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase from Escherichia coli was irreversibly inactivated on exposure to the affinity analog 2`,3`-dialdehyde ATP (oATP). The reaction displayed complex saturation kinetics with respect to oATP with an apparent K of approximately 0.8 mM. Reaction with radioactive oATP demonstrated that complete inactivation of the enzyme corresponded to reaction at two or more sites with limiting stoichiometries of approximately 0.7 and 1.3 mol of oATP incorporated/mol of PRPP synthetase subunit. oATP served as a substrate in the presence of ribose-5-phosphate, and the enzyme could be protected against inactivation by ADP or ATP. Isolation of radioactive peptides from the enzyme modified with radioactive oATP, followed by automated Edman sequencing allowed identification of Lys, Lys, and Lys as probable sites of reaction with the analog. Cysteine 229 may also be labeled by oATP. Of these four residues, Lys is completely conserved within the family of PRPP synthetases, and Lys is found at a position in the sequence where the cognate amino acid (Asp) in human isozyme I PRPP synthetase has been previously implicated in the regulation of enzymatic activity. These results imply a functional role for at least two of the identified amino acid residues.


INTRODUCTION

5-Phosphoribosyl-alpha-1-pyrophosphate (PRPP) (^1)synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase EC 2.7.6.1)is a paradigm of the small subclass of nucleotide-utilizing enzymes catalyzing reactions involving nucleophilic attack at the beta-phosphoryl group of the nucleoside triphosphate chain(1, 2, 3) . The product PRPP is an important precursor in several major biosynthetic pathways, being utilized in de novo nucleotide biosynthesis, nucleobase salvage, pyridine coenzyme production, and in plants and microorganisms, the biosynthesis of tryptophan and histidine(4, 30) . Thus, a detailed understanding of the mechanism of catalysis and the regulation of this enzyme is important from both an enzymological and physiological point of view: The enzyme serves as a ``mechanistic bridge'' between the far more common enzymes that catalyze nucleophilic attack at the alpha- and -phosphates (e.g. nucleotidyltransferases and kinases, respectively), and PRPP is an important metabolite, and thus a potential control point, in intermediary metabolism.

The reaction catalyzed by PRPP synthetase is ATP + Rib-5-P PRPP + AMP. The enzyme requires the presence of divalent metal ions for activity, with the preferred cation being Mg(5, 6) . Furthermore, Escherichia coli and Salmonella typhimurium PRPP synthetases are inhibited by ADP through binding at the active site and at an allosteric site specific for this nucleotide(7, 8, 9) . In spite of the fact that PRPP synthetases from several diverse species have been cloned and sequenced (e.g.(10, 11, 12, 13, 14) ), and that some of these have been purified to homogeneity and characterized(9, 15, 16, 17, 18) , there exists surprisingly little information about the nature of individual amino acids present in the active site of PRPP synthetase and how they participate in catalysis or substrate binding. Even less is known about the types of amino acid residues present in the allosteric site and their role in the control of enzymatic activity. In the absence of a high resolution three-dimensional structure, identification of functionally important residues has been limited to indirect methods such as chemical modification, mutagenesis, genetic methods, and comparative sequence analysis. Affinity labeling studies with 5`-p-fluorosulfonylbenzoyladenosine(19) , mutagenesis studies(20) , and characterization of mutants isolated by genetic methods (21) have led to the identification of a region of highly conserved amino acid sequence within the PRPP synthetase family. This region may be involved in ATP or divalent cation binding and includes a histidyl residue that may act as a general base during catalysis. Sequence comparisons with other proteins in the data base(9) , in combination with mutagenesis studies(22, 23) , have identified another conserved region possessing similarity to the phosphoribosyltransferases and containing conserved aspartyl residues which may be involved in Rib-5-P or PRPP binding.

Chemical modification studies using the group-specific reagent pyridoxal-5-phosphate implicated one or more lysyl residues as being functionally important in S. typhimurium PRPP synthetase. Pyridoxal-5-phosphate was postulated to react at the active site of the enzyme near the Rib-5-P-binding site(24) . The affinity analog prepared by treating ATP with sodium periodate, oATP(25) , presents an attractive opportunity to identify this putative functional lysyl residue because the reagent contains the same reactive moiety as pyridoxal-5-phosphate and should be directed to the active site of the enzyme by virtue of its similarity to the natural substrate ATP. The reagent has previously been successfully used to identify lysyl residues in the active sites of several enzymes(26, 27, 28) . Furthermore, it is easily prepared, and any nucleotide with free 2`,3`-hydroxyls can be prepared in the same manner. This will allow use of the periodate derivative of the allosteric effector ADP to probe the allosteric site of PRPP synthetase.

We report here the identification of active-site residues through affinity labeling studies utilizing oATP and E. coli PRPP synthetase.


EXPERIMENTAL PROCEDURES

Materials

All buffers, solvents, and reagents were the best grade obtainable. AspN and Staphylococcus aureus V8 proteases were obtained from Boehringer Mannheim, TPCK-treated trypsin was obtained from Sigma. [2,8-^3H]ATP, [2,8,5`-^3H]ATP, and [8-^14C]ATP were obtained from New England Nuclear. Liquid scintillation counting was performed using Ecolume scintillant from ICN Biomedicals, Inc.

Purification and Assay of PRPP Synthetase

PRPP synthetase was purified using established procedures (9) from E. coli strain S(udp) (29) harboring plasmid pHO11 which overproduces PRPP synthetase(12) . PRPP synthetase activity was assayed as described(30) . oATP-dependent formation of PRPP was measured by adapting an orotate phosphoribosyltransferase assay (31) in which production of orotodine-5`-monophosphate, and thus consumption of orotate, depends linearly on PRPP production. This assay contained 5 mM MgCl(2), 5 mM Rib-5-P, 0.3 mM ATP or oATP, 0.3 mM orotate, and 20 IU/ml orotate phosphoribosyltransferase in 50 mM potassium phosphate buffer, pH 7.5. The decrease in orotate concentration was measured spectrophotometrically at 295 nm. Protein concentration was determined by the bicinchoninic acid protein assay (32) using bovine serum albumin as a standard.

Preparation of oATP

oATP was synthesized by oxidation of ATP with sodium periodate and purified as described(25) . This was also the case when radiolabeled oATP was prepared. To remove traces of unreacted ATP, oATP was further purified by RP-HPLC on a 5-µm Spherisorb ODS-2 4.6 mm 12.5 cm column utilizing isocratic elution at 1 ml/min. The solvent system was 10 mM KH(2)PO(4) titrated to pH 5 with triethylamine. The quality of the separation of oATP from ATP was estimated by thin layer chromatography on polyethylenimine-cellulose plates developed in 0.85 M potassium phosphate, pH 3.4. The specific activity of radioactive oATP was determined by scintillation counting and determination of nucleotide concentration by UV spectroscopy utilizing an extinction coefficient of 15,400 M cm.

Reaction of oATP with PRPP Synthetase

PRPP synthetase was incubated with oATP at 30 °C for 3-5 h at oATP concentrations of 1-2 mM. The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.5, and 5 mM MgCl(2). Other specific additions are noted in the text. The enzyme concentration was 1 mg/ml (29 µM) in the reaction mixture unless otherwise indicated. Free oATP was separated from the oATP enzyme adduct using the spun column technique of Penefsky(33) . These columns were constructed in 1-ml syringes by centrifuging Sephadex G-25 equilibrated in 50 mM potassium phosphate buffer, pH 7.5, for 2 min at 5,000 g. At given times 40-100 µl was withdrawn from the reaction mixture, loaded on a column, and centrifuged as above. Using this technique 98% of unincorporated radioactivity was retained on the column as judged by subjecting pure [2,8-^3H]oATP to the procedure.

Reductive Alkylation of PRPP Synthetase

PRPP synthetase was dissolved in 10 mM NH(4)HCO(3) buffer, pH 7.5, and guanidine HCl was added to a final concentration of 6 M. Free sulfhydryl groups were reduced with dithiothreitol for 1 h and alkylated using 4-vinylpyridine for 1 h at room temperature as described previously(19) .

Proteolysis of [^3H]oATP-modified and -unmodified Enzyme

V8 protease was prepared as a stock solution of 5 mg/ml enzyme in 10 mM NH(4)HCO(3). Digestion was carried out at a final protease to substrate mass ratio of 1:25 in 10 mM NH(4)HCO(3) and 5% (v/v) CH(3)CN at 37 °C for 4-16 h. Digestion was monitored by RP-HPLC as described below. TPCK-treated trypsin and AspN protease were prepared as stock solutions of 1 mg/ml enzyme in 10 mM NH(4)HCO(3). The reaction conditions described above for V8 protease were also employed for trypsin and AspN protease digestions.

Peptide Chromatography

RP-HPLC was performed on a Waters chromatograph consisting of a model 660 controller and two model 510 pumps modified with microflow controllers. The solvent system consisted of 0.1% trifluoroacetic acid and 5% CH(3)CN as the A solvent, and 0.1% trifluoroacetic acid and 90% CH(3)CN as the B solvent. The gradient profile is indicated in the respective figure. Chromatographic separations were performed on 15 cm 2.1 mm narrow bore columns containing 5 µm C(18) silica-based chromatographic supports with 300-Å pores. These supports were obtained from Vydac and Shandon Scientific and packed according to the manufacturers' recommendations.

Automated Edman Sequencing and Amino Acid Analysis

Automated N-terminal amino acid sequencing was performed in an Applied Biosystems 477A protein sequencer using standard sequencing procedures as recommended by the manufacturer. Amino acid residues were identified as the phenylthiohydantoin (PTH) derivatives by on line RP-HPLC with comparison to known standards. When radioactive samples were sequenced, fractions from each sequencing cycle were collected and analyzed for radioactivity by liquid scintillation counting. Amino acid analysis was performed according to Barkholt and Jensen(34) .


RESULTS

Analysis of the Reaction of oATP with PRPP Synthetase

Incubation of PRPP synthetase with oATP resulted in specific inactivation of the enzyme. After 4 h of reaction with 2 mM oATP, the residual activity was 10-20% relative to that of the enzyme incubated under the same conditions but without oATP present. The activity of the control was stable over the course of the experiment. Fig. 1A shows a typical plot of the loss of activity with respect to time. The biphasic nature of the plot suggests that at least two reactions, one faster than the other, are occurring. The rate of reaction of the enzyme with oATP for both the slow and the fast reaction displays saturation kinetics (data not shown). Despite the complex reaction kinetics, pseudo-first-order rate constants, k, were estimated at different ATP concentrations from the average value of the rate constants for the fast and the slow portions of the overall reaction. The inset shows the double-reciprocal plot of kversus oATP concentration. Extrapolation yields an apparent K of approximately 0.8 mM.


Figure 1: Kinetics and stoichiometry of the reaction of oATP with PRPP synthetase. Reaction conditions were as described under ``Experimental Procedures.'' At defined time points, samples were removed from the reaction mix and subjected to the spun column technique as described under ``Experimental Procedures.'' The resulting eluate fractions were assayed for enzymatic activity, protein concentration and, in stoichiometric determinations, radioactivity. A, the time course of the reaction in the presence of 2 mM oATP. The inset shows the double-reciprocal plot of the pseudo-first-order rate constants for the reaction of PRPP synthetase with varying concentrations of oATP. The pseudo-first-order rate constant, k, was calculated as an average value consisting of the rate constants for the fast reaction and the slow reactions. The arrow indicates the value at which the apparent K was determined. B, the stoichiometry of inactivation of PRPP synthetase with [2,8-^3H]oATP.



The stoichiometry of the inactivation of PRPP synthetase was determined using [8-^14C]oATP or [2,8-^3H]oATP. Fig. 1B shows the relation between inactivation of the enzyme and incorporation of the analog. Again, the biphasic form of the curve suggests two or more heterogeneous reaction sites. Extrapolation of the data to complete inactivation for the first site of reaction yielded a value of about 0.7 mol of oATP incorporated/mol subunit of the enzyme. Extrapolation of the second site of reaction yields a value of about 1.3 mol of oATP incorporated/mol subunit. The stoichiometry of inactivation was somewhat variable and ranged from 0.5 to 0.8 for the first site and 1.1 to 1.5 for the second site.

The enzyme was protected from inactivation and incorporation of radioactive oATP when ATP was present in the reaction mix. Table 1shows the residual activity of PRPP synthetase after 3 h of reaction with oATP when varying concentrations of ATP were present in the mixture from the beginning. ADP at similar concentrations was also able to protect the enzyme against inactivation with oATP (data not shown). The latter agrees well with the fact that ADP can function as a competitive inhibitor with respect to ATP(7, 8) .



To bolster the argument that oATP binds and reacts at the active site, oATP was tested as a substrate for the PRPP synthetase reaction. oATP is altered in the ribose moiety of ATP, but the phosphate groups of oATP are probably intact. The enzyme activity was assayed using the coupled assay described under ``Experimental Procedures.'' PRPP was consistently formed in molar amounts that were equivalent to the utilization of about 50% of the added oATP when used as a pyrophosphoryl donor. The reaction was at least an order of magnitude slower than the reaction utilizing ATP as a substrate. No PRPP was formed in the absence of ATP or oATP. oATP has previously been shown to act as a substrate in several enzymatic reactions(26, 28, 35) .

The results of the kinetic, stoichiometric, and protection studies discussed above, combined with the fact that E. coli PRPP synthetase can use oATP as a substrate are compelling evidence that oATP is functioning as an ATP analog and reacting in a limited fashion at the active site of the enzyme. This is further supported by the proteolytic analyses of oATP-modified enzyme discussed below.

Amino Acid Analysis of [2,8-^3H]oATP-modified and -unmodified PRPP Synthetase

Both unmodified enzyme and enzyme modified with radioactive [2,8-^3H]oATP (1.3 mol of oATP bound/mol of PRPP synthetase subunit) were reductively alkylated and extensively dialyzed against 50 mM NH(4)HCO(3). Under these conditions there was no loss of radioactive label from the [2,8-^3H]oATP-modified enzyme preparation. This indicated that the modification was stable to the conditions of alkylation and dialysis, both of which were performed at slightly alkaline pH (approx8). The stability of the modification to these conditions was not influenced by whether or not the [2,8-^3H]oATP-modified enzyme was treated with NaBH(4) prior to alkylation and reduction. Amino acid composition analysis failed to detect any difference in the lysine content between the modified and unmodified enzyme, nor was there any indication of any unidentified peaks in the elution profile of the amino acid analysis. These results suggest that the oATP-enzyme adduct is labile under the acidic conditions of hydrolysis used for the analysis. This, in combination with the fact that borohydride reduction did not influence stability implies that oATP reacts with PRPP synthetase to yield a product other than a Schiff's base.

Isolation of Radiolabeled Peptides from [^3H]oATP-modified PRPP Synthetase

PRPP synthetase was labeled with 2 mM [^3H]oATP for 4 h, reductively alkylated, and extensively dialyzed as described under ``Experimental Procedures.'' The resulting labeled enzyme, which contained 1.4 mol oATP/mol subunit, was digested with TPCK-treated trypsin and chromatographed as described. The column eluant was monitored at 220 nm and the elution profile is shown in Fig. 2A. Fractions were collected during this chromatographic separation and assayed for radioactivity to generate the radioactivity profile shown by the closed circles in Fig. 2B. Two peaks are observed: peak 1, corresponding to a retention time of 40 min, and peak 2corresponding to a retention time of 44 min. The two peaks of radioactivity correspond to the peaks designated 1 and 2 in the UV profile shown in Fig. 2A. It should be noted that undigested, [^3H]oATP-labeled PRPP synthetase elutes at 62 min in this system, well away from peaks 1 and 2. Peak 1 elutes in a crowded region of the UV elution profile and appears to be composed of multiple peaks, whereas peak 2 is quite well resolved. Upon rechromatography of these peaks in the same system, but utilizing a shallower gradient, peak 1 is resolved into two radioactive peaks, peak 1A and peak 1B. In contrast, rechromatography of peak 2 still yields a single radioactive peak (data not shown). Thus, the major radioactive peaks shown by the closed circles in Fig. 2B result from three radioactive species, designated peak 1A, peak 1B and peak 2.


Figure 2: Chromatographic analysis and isolation of radioactive tryptic peptides from [^3H]oATP modified PRPP synthetase. The modified enzyme was prepared by labeling 20 mg of PRPP synthetase at a concentration of 1 mg/ml with 2 mM [^3H]oATP as described under ``Experimental Procedures.'' A, the UV elution profile at 220 nm of a 500-µg sample of tryptic peptides from [^3H]oATP-labeled PRPP synthetase chromatographed as described under ``Experimental Procedures.'' The gradient profile is indicated and consists of an initial hold at 5% CH(3)CN followed by a linear increase to 47.5% CH(3)CN, then followed by a final increase to 90% CH(3)CN with return thereafter to initial conditions. Fractions of 400 µl were collected. For panels B and C, 50 µl of each 400-µl fraction was assayed for radioactivity. The numbers in panel A correspond to the peaks with associated radioactivity in panels B and C. B, (bullet): the profile of radioactivity obtained from chromatography of tryptic peptides resulting from 4 h of treatment of [^3H]oATP-modified enzyme with TPCK-treated trypsin; (): the profile of radioactivity obtained from chromatography of tryptic peptides resulting from PRPP synthetase modified with [^3H]oATP in the presence of 4 mM ATP. C, change in distribution of radioactivity from chromatography of tryptic peptides isolated from a similar preparation of [^3H]oATP-modified PRPP synthetase as described above, but which had been digested with TPCK-treated trypsin overnight (geq12 h) This preparation had a similar UV elution profile to that shown in panel A.



To investigate whether the appearance of these radioactive peaks was due to specific labeling by oATP at the ATP-binding site, the enzyme was incubated with [^3H]oATP under the same conditions as described above, but in the presence of 4 mM ATP. The radioactivity profile shown by the open circles in Fig. 2B clearly demonstrates that all sites are protected against reaction with oATP when ATP is present. This strongly supports the results obtained from the reaction kinetics, stoichiometry, and protection studies demonstrating that oATP functions as an ATP affinity analog for PRPP synthetase.

The pattern of radioactivity observed in Fig. 2B (closed circles) was somewhat variable. The relative distribution of radioactivity between peaks 1 and 2 appears to depend on several factors. Fig. 2C shows the effect of extended TPCK-treated trypsin digestion (geq12 h) on a similar preparation of [^3H]oATP-labeled enzyme. A new peak, peak 3, which elutes at the column void volume is observed. In addition, a similar peak is observed in chromatographic separations of peptides derived from digestions with AspN and V8 proteases (data not shown). Peak 3 may be a breakdown product of the oATP-enzyme adduct. This breakdown product may be generated by chromatography of peptides derived from the modified enzyme in the acidic trifluoroacetic acid/CH(3)CN solvent system, or by extended incubation times at 37 °C during proteolysis. That breakdown was the cause of the appearance of peak 3 is based upon four observations: (i) the radioactivity profile of pure [^3H]oATP chromatographed in this system yields a peak that also elutes at the void volume; any breakdown product might be expected to have similar chromatographic properties to authentic oATP in this system; (ii) rechromatography of pure, undigested [^3H]oATP-labeled PRPP synthetase previously isolated by chromatography in this system yields two peaks: the undigested, labeled PRPP synthetase subunit eluting at 62 min, and a peak eluting at the void volume; (iii) rechromatography of peaks 1 and 2 yields radioactive peaks eluting at the void volume; and (iv) amino acid analysis indicates that the oATP-enzyme adduct is unstable when exposed to acidic conditions. Alternatively, peak 3 may be a very small, oATP-labeled peptide not retained on the C(18) column. In the case of trypsin, this peptide could be generated from slow cleavage at an oATP-modified lysyl residue. Thus, this peak would appear only after extended treatments with trypsin. Breakdown of oATP-peptide adducts has been previously reported(26, 36) , while the results of our sequencing studies discussed below suggest that slow tryptic cleavage at a modified lysyl residue may occur. It is, however, more likely that breakdown is the largest factor contributing to the appearance of peak 3 in tryptic digests of [^3H]oATP-labeled PRPP synthetase, given the results with other proteolytic digestions, and with the undigested, labeled enzyme.

Identification of the Sites of Reaction of oATP with PRPP Synthetase

In order to identify the residues labeled by reaction of oATP with the enzyme, amino acid sequencing of the major radioactive peaks (1A, 1B, and 2) from the tryptic digestion was undertaken. Rechromatographed radioactive peptides were subjected to automated Edman degradation as described. A summary of these results is shown in Table 2.



The isolated radioactive peptides contained sequences that include lysines 181, 193, and 230 (see Fig. 3). The sequence of peptide 2 overlapped the sequence of the peptide 1B. This could be the result of slow cleavage at a modified lysyl residue (Lys) as described in the previous section or, alternatively, partial modification of Lys. It is important to note that, regardless of the peptide sequenced, significant radioactivity was only found associated with the PTH fraction from the first sequencing cycle of each peptide. There was very little radioactivity retained on the sequencing filter and none detected in the waste effluent from the sequencer. Thus, it is likely that the radioactivity in the oATP-peptide adduct is lost under the relatively harsh conditions encountered during the sequencing cycle. The mechanism of this breakdown is not known, but several breakdown species are possible. Radioactive isotope is present in the purine ring of the analog, and the purine ring can be lost under acidic or basic conditions. Furthermore, the chromatogram from the PTH analysis of cycle 1 of each peptide revealed the presence of a UV absorbing peak ( = 269 nm) eluting closely to PTH-Asp. This peak was present in anomalously large amounts and was not present when peptides isolated from unmodified PRPP synthetase were sequenced. These results are consistent with the suggestion that this UV absorbing peak is derived from the purine ring or adenosyl portion of the oATP-enzyme adduct. Finally, trace amounts of radioactivity (3-5% of the total radioactivity in the isolated peptide applied to the sequenator) could be detected in the PTH fractions corresponding to the modified lysines when very high specific activity oATP (5.5 10^8 disintegrations/min/µmol) was used to label PRPP synthetase. Further evidence that these lysyl residues are the sites of modification is provided by comparison of the yield of PTH lysine derivatives present at suspected modification sites to the overall yield of the other PTH-derivatives present in the sequence. The amounts of the PTH-lysine at these sites (shown in boldface in Table 2) were reduced 50-100% when compared to the expected yield based upon the PTH-derivatives in the previous cycles. Recovery of the final PTH-derivative at the end of peptides is often low, but a significant increase in yield at this position is very unusual. In peptides 1B and 2, recovery of the the final arginines increase by 50 and 30%, respectively. Thus, it is likely that this increase represents an anomalously low yield in the preceding cycle, consistent with assignment of the modification of Lys at that position. Firm assignment of Lys as a site of labeling in peptide 1A was more problematic. The absence of an identifiable PTH-derivative at the position corresponding to Cys could be because of poor recovery of peCys, or modification at this site. These residues are well in from the N terminus of the peptide at a position where the sequence signal begins to decay, and no readable sequence was detected after Lys in peptide 1A. Alternatively, cross-linking by oATP of adjacent lysine and cysteine residues has previously been postulated(36) .


Figure 3: Alignment of PRPP synthetase amino acid sequences in the region of oATP labeling. Numbers indicate the amino acid from the N terminus of the polypeptide in the cases of the E. coli(9) , S. typhimurium(10, 41) , B. subtilis(13, 15) , and human (16, 42) enzymes. The other proteins are numbered from the initiator methionines of the amino acid sequence deduced from the respective DNA or cDNA sequence. The magenta lysyl residues in the E. coli sequence indicate the sites of reaction with oATP. The blue lysyl residues in the other sequences show lysyl residues that may be equivalent to Lys in E. coli PRPP synthetase. The green residues in the E. coli sequence show the region identified as a putative PRPP-binding site based upon homology to the phosphoribosyltransferases(9) . Asterisks above amino acids indicate identical residues among the sequences. Segments 1-3 correspond to segments 1, 2, and 3, respectively, of sequences suggested to be involved in ATP binding in adenylate kinases and related proteins(14) . Vertical boxes indicate the amino acids in the other enzymes that occupy homologous positions to the sites of labeling in the E. coli enzyme. The radioactive peptides isolated in this work are indicated by the labeled bars over the respective region of amino acid sequence. The Leishmania donovani cDNA sequence has been published(11) . The unpublished nucleotide sequences shown were retrieved from the GenBank/EMBL Data Bank and have the following accession numbers: Arabidopsis thaliana, X83764; Bacillus caldolyticas, X83708; Listeria monocytogenes, M92842; Caenorhabditis elegans, U00036; Synechococcus sp., D14994.



To strengthen the argument that Lys is labeled by oATP, and to investigate whether Cys was also labeled, isolation and sequencing of radioactive peptides from digestion of [^3H]oATP-labeled PRPP synthetase with AspN protease was undertaken. Sequence analysis of a radioactive AspN peptide containing Lys is also shown in Table 2. Inspection of the PTH-derivative yields indicates that the Lys position indeed shows a decreased yield of PTH-Lys, indicative of labeling at this position. While peCys was not quantitated, inspection of the chromatogram of this cycle shows that the yield of PTH-peCys is reduced only slightly (data not shown). This result implies that modification of Cys by oATP was either very minor, or that the adduct of oATP with cysteine, presumably a thiohemiacetal, is even more unstable to the sequencing conditions than the lysine adduct. An interesting side note was that this AspN peptide's C terminus was generated by cleavage before a glutamyl residue in the PRPP synthetase sequence. The manufacturer's product information sheet states that AspN protease cleaves at aspartyl residues 2000 times faster than at glutamyl residues.

The observation that these three lysyl residues would all be contained within a single large peptide generated from treatment of modified enzyme with S. aureus V8 protease under conditions where cleavage is restricted to glutamic acid residues (37) prompted us to examine V8 digests of modified enzyme by RP-HPLC. A single radioactive peptide accounting for 80-90% of the amount of radioactivity applied to the chromatograph was observed on RP-HPLC analysis of V8 digested [^3H]oATP-modified PRPP synthetase (data not shown). The rest of the radioactivity eluted at the column void volume. This result in combination with the results discussed above, supports our assignment of Lys, Lys, Lys as sites of reaction of the enzyme with oATP.


DISCUSSION

Our affinity labeling results localize the sites of reaction of oATP with PRPP synthetase to a region of sequence spanning approximately 60 amino acid residues. Fig. 3shows an alignment of this sequence with the homologous amino acid sequences of known PRPP synthetases isolated from several diverse species.

Two of the identified residues, Lys and Lys are especially noteworthy. Lysine 193 is completely conserved among all known PRPP synthetases. Only two lysyl residues are completely conserved in the PRPP synthetase family. Strong conservation of Lys across broad phylogenetic distances, in combination with our results indicating that it lies in the active site, implies that the residue possesses an important function in the protein. Interestingly, this residue lies at the end of a stretch of sequence (segment 2, Fig. 3) that has similarity to one of three sequence segments found in adenylate kinases and other enzymes (14, 38) . The segment 2 consensus sequence for adenylate kinases is -K---X-K- where and X represent hydrophobic and any amino acid, respectively. From the crystal structure of adenylate kinase, the hydrophobic residues appear to form a binding pocket for the adenosyl moiety of MgATP, while NMR measurements indicate that the first lysine in the consensus sequence could interact with the beta- and/or -phosphate group of ATP(38) . Lysine 193 in the PRPP synthetases occupies the cognate position of the second lysine in the adenylate kinase consensus sequence. The function of the second lysine in the adenylate kinase segment 2 sequence has not been delineated. It is possible that Lys of E. coli PRPP sythetase interacts with ATP, perhaps by forming hydrogen bonds with either the 2`- or 3`-hydroxyl groups. This is an attractive idea because Lys reacts with the aldehyde functionalities in oATP that are present at positions analogous to the 2`,3`-hydroxyl groups of ATP. Alternatively, Lys could be interacting with the triphosphate chain of ATP either directly, or indirectly through the Mg cation chelated to the phosphate moieties in the MgATP complex utilized as a substrate by PRPP synthetase.

In contrast, Lys is conserved in only four of the PRPP synthetases. In all other PRPP synthetases, the residue at this position is an aspartic acid. Interestingly, there are relatively well conserved lysyl residues present either upstream, downstream, or both in the other sequences. Of even greater interest, however, is the fact that the aspartic acid residue (Asp) found at this position in human isozyme I is replaced by a histidyl residue in a mutant PRPP synthetase isolated from patient S. M.(39) . This mutant enzyme is characterized by greatly decreased sensitivity to inhibition induced by purine ribonucleoside diphosphates that act both competitively (ADP) and noncompetitively (ADP, GDP) with respect to ATP. This raises the intriguing possibility that the residue found at this position, or alternatively, the sequence around this residue is somehow involved in controlling inhibition of the enzyme. Kinetic inhibition studies with PRPP synthetase from S. typhimurium suggested the existence of a second, allosteric site for ADP binding (8, 9) . Equilibrium dialysis binding studies (7) confirmed that the second site existed and demonstrated that the allosteric site is available for ADP binding only in the presence of saturating amounts of the substrates ATP (or the nonhydrolyzable substrate analog, alpha,beta-methylene ATP) and Rib-5-P. It was furthermore shown that this site was highly specific for ADP and did not bind ATP. Our affinity labeling results with the E. coli enzyme imply that Lys is in the active site of the enzyme. Lysine 181 could thus be involved in transmitting a signal to the allosteric site from the active site when it binds substrates, with the result that the allosteric site becomes competent to bind inhibitor. Although lysine is replaced by aspartate at the homologous position in human isozyme I, there is a lysine residue six amino acid residues upstream of this position that could possibly carry out a similar function. Alternatively, it is conceivable that Asp in the human enzyme functions in a subtly different way than Lys does in the E. coli enzyme. It is interesting in this respect that PRPP synthetases from the enteric bacteria are insensitive to allosteric inhibition by GDP, whereas the mammalian and Bacillus subtilis PRPP synthetases are quite sensitive to GDP inhibition. These GDP-sensitive enzymes all possess aspartate residues at the cognate position of Lys in the E. coli enzyme. Thus, differences in the primary structure of PRPP synthetases in the region around Lys may play a role in determining this specificity. Finally, it is possible that this position represents a junction between the allosteric and the active sites, controlling inhibitor specificity and sensitivity by virtue of being shared between the two sites.

It is more difficult to draw conclusions concerning function about the site of labeling at Lys and, if it exists, Cys. These sites are protected from modification by oATP in the presence of ATP and could therefore lie in or near the active site. They are found just downstream of the highly conserved, putative PRPP-binding site (9) in a relatively well conserved region of sequence. Cysteine at this position is conserved in about 50% of the PRPP synthetases, while at the Lys position charged residues and leucine predominate; lysine is present in only four of the PRPP synthetases. Segment 3 (Fig. 3) of the adenylate kinase homologous sequences is also found within the PRPP-binding site sequence. Segment 3 has been postulated to interact with the triphosphate chain of ATP in these enzymes(38) . Previous studies with sulfhydryl specific reagents (40) identified Cys in S. typhimurium PRPP synthetase as being very reactive, but possessing no functional role with respect to substrate binding or catalysis. Although these considerations make it unlikely that Cys and Lys have a specific function in catalysis or ligand binding in E. coli PRPP synthetase, affinity labeling results could be explained by these residues lying at the entrance to the active site and being located in a region that alters conformation upon substrate binding, such that saturation with ATP prevents reaction with oATP.

The relationship of the identified lysyl residues in this study to the ``critical lysine'' implicated by pyridoxal phosphate inactivation studies of the S. typhimurium enzyme (24) is not clear. If one assumes that one of our lysines is this critical lysine, ATP protection studies also reported in this study indicating that ATP does not fully protect against inactivation are difficult to reconcile with our studies showing ATP prevents both inactivation and incorporation of oATP. The reason for the discrepancy may lie in the intrinsic differences between the reagents employed. The suggestion that this critical lysine lies in the Rib-5-P subsite may indicate that this residue is not represented among our labeled lysines. Elucidation of these details will require identification of the lysine residue labeled by pyridoxal phosphate.

Although oATP has been assumed to be specific for lysines, our work and the results of Zheng et al.(36) imply that cysteine may be capable of reacting with this reagent as well. The expected reaction product would be a thiohemiacetal. Zheng et al. also identified a cysteinyl residue adjacent to a lysyl residue where both appeared to be labeled, and they postulated that oATP cross-links these residues through reaction of both aldehyde functionalities present in oATP. A similar reaction might occur in our system. Furthermore, although it is often stated that oATP forms a Schiff's base with lysine, there is ample evidence in the literature that this is the exception rather than the rule(26, 35, 36) . Our observations on the lack of effect of reducing agent on the stability of the oATP-enzyme adduct as well as the instability of the adduct to acid pH support this contention.

Finally, ADP is an allosteric inhibitor of PRPP synthetase that binds at a site distinct from the active site, is specific for ADP, and access to which is influenced by substrate saturation of the active site. These circumstances make similar affinity labeling studies probing the allosteric site with oADP an attractive prospect. Studies with this aim are currently underway in our laboratory and may provide insight into the nature of amino acids present in the allosteric binding site. These studies, in combination with x-ray crystallographic studies of B. subtilis PRPP synthetase in progress in our laboratory should produce new insights into the molecular mechanism of catalysis and allostery in PRPP synthetase.


FOOTNOTES

*
This research was supported by funds from The Danish Natural Science Research Council (to I. H.) and The Danish Basic Research Foundation (to B. H.-J. and to K. W. H.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]; Bacillus caldolyticus, X83708[GenBank]; Listeria monocytogenes, M92842[GenBank]; Caenorhabditis elegans, U00036[GenBank]; Synechococcus sp., D14994[GenBank].

§
To whom correspondence should be addressed: Dept. of Protein Chemistry, University of Copenhagen, Farimagsgade 2A, DK-1353 Copenhagen K, Denmark. Tel.: +45-35-32-20-81; Fax: +45-35-32-20-75; kharlow{at}biobase.dk.

(^1)
The abbreviations used are: PRPP, 5-phosphoribosyl-alpha-1-pyrophosphate; oATP, 2`,3` dialdehyde ATP; Rib-5-P, ribose-5-phosphate; RP-HPLC, reverse phase high performance liquid chromatography; TPCK, tosylphenylchloromethylketone; peCys, S-pyridylethylcysteine; PTH, phenylthiohydantoin.


ACKNOWLEDGEMENTS

We gratefully acknowledge Martin Willemoës for his critical comments and the idea of using a coupled PRPP synthetase assay to measure PRPP formation from oATP and Drs. R. L. Switzer and Kaj Frank Jensen for their critical reading of the manuscript. We also thank Dr. Kaj Frank Jensen for graciously providing the orotate phosphoribosyltransferase used in this study.


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