Báo cáo khoa học: Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus

Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus Kaj F. Jensen1, Susan Arent2,*, Sine Larsen2,† and Lise Schack1

1 Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Denmark 2 Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Denmark

Keywords extremophiles; phosphoribosyl diphosphate, pyrimidine nucleotide biosynthesis; pyrimidine salvage; thermostable enzymes

Correspondence K. F. Jensen, Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83H, DK-1307 Copenhagen K, Denmark Fax: +45 35322040 Tel: +45 35322020 E-mail: kfj@mermaid.molbio.ku.dk

Present addresses *Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500, Denmark †European Synchrotron Radiation Facility, ESRF-BP 220, F-38043 Grenoble Cedex, France

(Received 2 November 2004, revised 18 January 2005, accepted 20 January 2005)

doi:10.1111/j.1742-4658.2005.04576.x

The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when assayed at 60 (cid:1)C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl- 1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP was complex as it depended on the presence of the reac- tion product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors CTP and UMP can be bound simultaneously (KD equal to 2 and 0.5 lm, respectively). The bind- ing of each of the inhibitors was incompatible with binding of PRPP or GTP. The data indicate that UPRTase undergoes a transition from a weakly active or inactive T-state, favored by binding of UMP and CTP, to an active R-state, favored by binding of GTP and PRPP.

[2–4], Toxoplasma gondii

hyperbolic kinetics for specific for de novo purine nucleotide synthesis, is feed- back inhibited by AMP and GMP, and uracil phos- phoribosyltransferase (UPRTase; a salvage enzyme that makes UMP from uracil and PRPP) was shown to be activated by GTP in several organisms e.g. Escherichia coli [5], Giardia intestinalis [6] and Sulfolobus shibatae [7], while its activity appears unregulated in other organisms such as yeast [8] and Bacillus caldolyticus [9,10].

Abbreviations IPTG, isopropyl thio-b-D-galactoside; GdHCl, guanidinium chloride; PPi, inorganic diphosphate or pyrophosphate; PRPP, 5-phosphoribosyl-1-a- diphosphate; UPRTase, uracil phosphoribosyltransferase or UMP synthase (EC 2.4.2.9).

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1440

The phosphoribosyltransferases catalyze the formation of nucleotides by reaction with 5-phosphoribosyl-1- diphosphate (PRPP) and a nitrogenous base, releasing pyrophosphate as the second reaction product. The enzymes participate in the biosynthesis of the amino acids histidine and tryptophan, pyridine coenzymes, and all nucleotides by de novo synthesis and salvage pathways [1]. They are generally unregulated enzymes obeying the saturation substrates, but there are exceptions. The glutamine PRPP amidotransferase, which catalyses the first step Crystal structures have been determined for the [5,11], UPRTases from B. caldolyticus [10], T. gondii

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

Fig. 1. SDS ⁄ PAGE illustrating the purification of UPRTase. M, molecular mass marker proteins of 97, 66, 45, 31 and 14 kDa; 1, crude extract; 2, supernatant after heating at 75 (cid:1)C; 3, redissolved pellet after ammonium sulfate precipitation; 4, pooled fractions from DEAE–cellulose column; 5, pooled fractions from Dye Matrex Green A column. The gel was stained with Coomassie Blue.

Thermotoga maritima [12] and E. coli (A. Kadziola, Center for Crystallographic Studies, University of Copenhagen, Denmark, pers comm). All UPRTases have a structure that displays a core five-stranded twis- ted b-sheet that binds the 5-phosphoribosyl moiety of PRPP and the nucleotide product. This fold is shared by other phosphoribosyltransferases [13] and PRPP synthase [14] but they can exist in different oligomer states. The PRPP binding motif is characteristic for the UPRTases [15]. Near the C-terminal end they contain a highly conserved sequence that is involved in uracil binding, but no sequence motif has been identified that separates the GTP-activated enzymes from the unregu- lated enzymes. The mechanism of activation of UPR- Tase activity by GTP was investigated for the E. coli and T. gondii enzymes [4,5] and was found to involve oligomerization of a dimeric enzyme with low activity to a more active tetrameric enzyme. The tetrameric form is stabilized by PRPP and ⁄ or GTP, both of which contact two dimers of the tetramer, and for this reason the stimulatory effect of GTP is only seen at subsatu- rating concentrations of PRPP [4,5].

(pLFS2) was constructed by cloning a PCR copy of the upp gene into plasmid pUHE23-2 [18] so that tran- scription was brought under the control of the lacI regulated PA1 ⁄ 04 ⁄ 03 promoter, as described in the Experimental procedures.

The enzyme from the archaeon Sulfolobus shibatae is unusual amongst the GTP-activated UPRTases as it is strongly inhibited by CTP, a feature not previously seen for any phosphoribosyltransferase [7]. The enzyme from S. shibatae was studied in a crude preparation. We were interested in characterizing the allosteric properties of the enzyme, but were unable to obtain it in a homogen- eous form or clone it by complementation in E. coli. For that reason we cloned the upp gene from the closely rela- ted organism Sulfolobus solfataricus, whose genome has been sequenced [16], to produce the enzyme in sufficient amounts to study its molecular properties. This paper describes studies of the kinetic behavior with emphasis on elucidating the mechanism of allosteric regulation. The results show that the activation by GTP results from an increase in the catalytic rate (kcat) and that it does not involve changes of the oligomeric state of the enzyme, which is a tetramer, regardless of the presence of substrates and effector molecules. Crystal structures of the enzyme in complex with CTP and ⁄ or UMP are described elsewhere [17].

Results

Cloning, expression and purification The enzyme was purified from strain NF1830 trans- formed with pLFS2 and grown under inducing condi- tions. UPRTase constituted 2–3% of the total cellular protein. The protein was purified to near homogeneity by a procedure that included heating at 75 (cid:1)C, ammo- nium sulfate precipitation, column chromatography on DEAE–cellulose, Dyematrex Gel Green A and Sephacryl G-200 (Fig. 1). The purified protein was approximately 85% saturated with UMP (Fig. 2). The bound ligand could be removed by gel-filtration chromatography of the unfolded protein on a SephadexTM G-25 column in the presence of 6 m guanidinium chloride (Fig. 2B). Dialysis against 10 mm Tris ⁄ HCl, 0.1 mm EDTA, pH 8, resulted in refolding and recovery of about two thirds of the enzyme activity. The rest of the enzyme precipitated and was removed by centrifugation. The refolded enzyme behaved as the native UMP saturated protein in kinetic experiments and was primarily used for studies of ligand binding and sedimentation behavior.

Optimum pH

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1441

The upp gene encoding the UPRTase of S. solfataricus was identified as the open reading frame SSO0231 in the genomic sequence [16]. The protein was predicted to contain 216 amino acid residues and have a relative molecular mass of 24.1 kDa. An expression vector The rate of UMP synthesis in the interval pH 3–8 was determined at 60 (cid:1)C in the presence of uracil

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

Fig. 3. Activation of UPRTase activity by GTP. The curves show the activity of UPRTase as a function of GTP concentration. The PRPP concentration was 1 mM, otherwise conditions were as stated in Experimental procedures. s, no further addition; e, UPRTase in the presence of 1.1 mM UMP.

maximal activity in the pH range 5.0–5.6 and pH 5.5 was chosen for all further analyses. The pH activity profile (not shown) was asymmetric, due to a more rapid decline of activity on the acidic side of the opti- mum than on the alkaline side. Activity was essentially absent at pH 3.8, whereas approximately 20% of the activity remained at pH 8.

Effects of allosteric ligands on the kinetics of UPRTase

Fig. 2. UMP content of purified UPRTase. (A) UMP content analyzed by acid precipitation of the protein. The absorbance spectrum of the protein before the addition of acid is shown as a solid line (A280 ¼ 0.781). The dotted curve represents the spectrum of the acid super- natant (corrected for the 10% dilution) following removal of the preci- pitated protein by centrifugation. The absorption, A261 ¼ 0.341, indicated 34 lM UMP and therefore that (cid:1) 20% of the A280 of the enzyme sample stems from UMP at neutral pH. (B) Absorbance spectra from the gel-filtration analysis of unfolded protein in the pres- ence 6 M GdHCl (corrected to equal volume). Solid line, spectrum of protein in 6 M GdHCl before gel-filtration, A280 ¼ 0.903. Dashed line, spectrum of the eluted macromolecule. A280 ¼ 0.706 corresponds to 46 lM protein as the absorption coefficient A280 calculated from the amino acid composition would be 0.634 mg)1ÆmL)1Æcm)1 [19]. Dot- ted line, spectrum of eluted small molecules. The absorbance, A261 ¼ 0.390 and A280 ¼ 0.186, corresponds to 39 lM UMP indica- ting that the isolated enzyme represented (cid:1) 85% of this value. The identification of UMP as the nucleotide bound to UPRTase is based on the UV light absorption spectra and the finding of UMP in the act- ive sites of UPRTase by X-ray crystallography [17].

Figure 3 shows that the rate of synthesis of UMP cata- lyzed by the UPRTase from S. solfataricus is strongly stimulated ((cid:1) 20-fold) by GTP. The activation curve is sigmoidal (nH ¼ 2.5 ± 0.3) and the concentration of GTP needed to give half maximal stimulation is 97 ± 5 lm. The presence of 1 mm UMP in the reac- tion mixtures made the cooperativity (nH ¼ 3.1 ± 0.3) of the GTP activation profile very evident and reduced the maximal reaction velocity, probably due to compe- tition with the substrate PRPP (Fig. 3).

lines,

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1442

(0.116 mm), PRPP (0.6 mm), Mg2+ (10 mm) and GTP (1 mm) using the buffers described in Experimental procedures to control the pH. The enzyme exhibited The initial velocity patterns for the UPRTase reac- tion in the presence or absence of GTP are shown in Fig. 4 in the form of double reciprocal plots. A pattern of parallel indicative for a ping-pong kinetic mechanism, was observed in the presence of GTP (Fig. 4A), while a pattern of intersecting lines, indicat- ive for a sequential mechanism, was observed in the

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

Fig. 4. Initial reaction velocities of the UPRTase reaction at varying concentrations of PRPP and uracil shown as double reciprocal plots. The buffer was 50 mM succinate ⁄ phosphate, pH 5.5, 10 mM MgCl2, with a reaction temperature of 60 (cid:1)C. Concentrations of PRPP were as indi- cated on the figure. (A) Kinetics of the reaction in the presence of 1 mM GTP, using an enzyme concentration of 275 ngÆmL)1. Uracil concen- trations were: s, 50 lM; h, 25 lM, e, 12.5 lM; n, 6.3 lM, ,, 3.1 lM. Fitting to the equation for a ping-pong reaction (Eqn 4) gave Vmax ¼ 15.3 ± 0.9 lmolÆmin)1Æmg)1, Km,PRPP ¼ 48 ± 7 lM and Km,uracil ¼ 25 ± 3 lM, while fitting to the rate equation for a sequential mechanism (Eqn 3) gave Vmax ¼ 15.8 ± 1.2 lmolÆmin)1Æmg)1, Km,PRPP ¼ 55 ± 11 lM, Km,uracil ¼ 27 ± 4 lM and the kinetic dissociation constant for the first substrate to bind Kia ¼ )5.9 ± 6.5 lM, not significantly different from zero. (B) Kinetics of the unactivated reaction, using an enzyme concentration of 550 ngÆmL)1. Uracil concentrations were twofold lower than in (A). Fitting to the rate equation for a sequential reaction mechanism (Eqn 3) gave Vmax ¼ 0.92 ± 0.07 lmolÆmin)1Æmg)1, Km,PRPP ¼ 19 ± 5 lM, Km,uracil ¼ 1.4 ± 0.4 lM and Kia ¼ 51 ± 31 lM. Fitting to the equation for a ping-pong reaction (Eqn 4) gave Vmax ¼ 0.96 ± 0.06 lmolÆmin)1Æmg)1, Km,PRPP ¼ 31 ± 5 lM and Km,uracil ¼ 2.3 ± 0.4 lM, but the lines, derived from this fit, deviated systematically from the data points.

absence of GTP (Fig. 4B). The kinetic constants derived from the data are given in the legend to Fig. 4, and it is apparent that the presence of GTP (1 mm) increases the turnover number (kcat) from 0.36 s)1 for the unactivated enzyme to 6.1 s)1 for the GTP-activa- ted enzyme and elevates the Km values for PRPP and uracil by two- and >10-fold, respectively.

We tested whether the UPRTase from S. solfataricus used the energy from GTP hydrolysis to drive the reaction forward and speed up the synthesis of UMP. This was carried out by making a set of reactions con- taining [32P]GTP[aP] at concentrations equal to 50, 100, 250 and 500 lm. Aliquots, sampled at different times during the reaction, were analyzed by chroma- tography on ion exchange thin-layer plates and no degradation of GTP was detected (not shown).

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1443

ATP and UTP (1 mm) had no stimulatory effects on UPRTase. Nor did these nucleotides (in concentrations up to 0.8 mm) influence the UPRTase activity meas- ured in the presence of 0.2 mm GTP. However, the activity was strongly inhibited by CTP, both in the presence and the absence of GTP. The inhibitory effect of CTP was complex because reactions containing CTP gradually came to a complete stop long before the substrates were used up, while reactions free of CTP continued for extended periods of time (Fig. 5A). We ascribe the declining reaction rate in the presence of CTP to the accumulation of UMP in the mixtures, as UMP became a very strong inhibitor in the presence of CTP. Figure 5B shows the time course of reactions that contained different concentrations of UMP from the start, without or with 0.5 mm CTP present. All reactions devoid of CTP continued as linear functions of time over the entire assay period (10 min) and the presence of 50 lm UMP only reduced the reaction velocity by approximately 15%. However, UMP inhib- ited the initial reaction rate very strongly in the pres- ence of CTP. The initial reaction velocity was close to zero when both CTP (0.5 mm) and UMP (50 lm) were present from the start of the reaction. It is also appar- ent from Fig. 5 that CTP only inhibits the reactions (with 1 mm PRPP and 1 mm GTP) weakly if at all in the absence of UMP, as the initial velocities of the reactions free of UMP were nearly identical with and without 0.5 mm CTP. The inhibitory effect of CTP became substantially stronger when the concentration of GTP was reduced from 1 mm to 0.2 mm, indicating a competition between GTP activation and CTP

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

immediately when an additional 0.5 mm GTP was added (not shown).

Reverse and exchange reactions

inhibition (Fig. 5C). Consistent with this observation, the presence of 0.5 mm CTP increased the concentra- tion of GTP needed to give half maximal reaction rate from (cid:1) 0.1 mm in the absence of CTP to 1.0–1.6 mm (not shown). Moreover, the CTP-containing reactions, which had ‘autoterminated’ after a short while (i.e. reactions similar to 2 and 4 in Fig. 5A), resumed

rate of at a

Fig. 5. Inhibition by CTP. (A) Time course of four reactions all of which contained 1 mM PRPP and 116 lM [14C]uracil. Reactions 1 and 3 were free of CTP while reactions 2 and 4 contained 1 mM CTP. In addition, reactions 1 and 2 contained 1 mM GTP while reac- tions 3 and 4 were free of GTP. Other components and tempera- the standard assay. Reaction 1 ture were as described for accumulated UMP as a linear function of time (v ¼ 2.2 lMÆmin)1) almost until all uracil was converted to UMP. In reaction 2, UMP accumulated to reach a concentration of (cid:1) 12 lM within 10–12 min after which it stopped almost completely. Reaction 3 without any allosteric effectors resulted in a linear accumulation of UMP as a function of time (v ¼ 0.9 lMÆmin)1) over the entire assay period, while reaction 4 accumulated UMP at a concentration (cid:1) 1.3 lM over a period of (cid:1) 25 min and appeared to terminate thereafter. (B) Combined inhibitory effects of CTP and UMP on the UPRTase reac- tion that contained 1 mM GTP, 1 mM PRPP and 116 lM uracil. The reactions contained different concentrations of UMP in the absence (filled symbols and crosses) or in the presence of 0.5 mM CTP (open symbols). The (initial) concentrations of UMP were diamonds, no UMP; n (10 lM); · (20 lM) and circles (50 lM). The reactions without CTP continued as a linear function of time over the assay period (10 min), but for clarity only the initial parts of the reactions with no UMP and 50 lM UMP are shown. (C) Influence of GTP concentration on the inhibitory effect of CTP. The reactions con- tained 1 mM PRPP, 116 lM uracil and either 1 mM GTP (s) or 0.2 mM GTP (n). CTP concentrations are indicated, while other con- ditions were as described for the standard assay.

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1444

The kinetics of the reverse reaction (i.e. the formation of PRPP and uracil from UMP and pyrophosphate) could not be assessed because the equilibrium strongly favors nucleotide formation (Keq (cid:1) 80 000 [15]) and in an exchange reaction containing [32P]PPi (0.1 mm) to PRPP (1 mm) absolutely no formation of [32P]PRPP could be detected, either in the presence or absence of 1 mm GTP. However, when 0.1 mm UMP was included in [32P]PRPP was formed at a rate of such a reaction, 0.03 lmolÆmin)1Æ(mg enzyme))1 in the absence of GTP, at a rate of 0.09 lmolÆmin)1Æ(mg enzyme))1 in the presence of 1 mm GTP and at a rate of 0.002 lmolÆmin)1Æ(mg enzyme))1 in the presence of 0.5 mm 14C radioactivity CTP. Likewise, no exchange of between uracil and UMP could be detected unless a third substrate PRPP or PPi was present. When the enzyme was incubated with [14C]UMP (50 lm) and ura- cil (0.1 mm) in the presence of 0.1 mm PPi, [14C]uracil 0.09 lmolÆmin)1Æ(mg accumulated enzyme))1. The rate was stimulated to 0.43 lmolÆ min)1Æ(mg enzyme))1 by the addition of 1 mm GTP and inhibited to an undetectably low level in the presence of 0.5 mm CTP.

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

Ligand binding

sigmoidal (nH ¼ 4). In the presence of 100 lm CTP no significant binding of PRPP at concentrations up to 200 lm could be detected (not shown). Figure 6B shows that the substrate PRPP stimulates binding of GTP, and inhibits binding of CTP.

Fig. 6. Ligand binding experiments. (A) Binding of [32P]PRPP to UMP-free UPRTase (26 lM). s, no further addition; e, in the presence of 1 mM GTP; ·, in the presence of 50 lM CTP. (B) Effect of PRPP on the binding of allosteric effectors to UMP-free UPRTase (19 lM). s, GTP alone; h, GTP binding in the presence of 1 mM PRPP; e, CTP alone; ·, CTP binding in the presence of 1 mM PRPP. (C) Binding of allosteric effectors to the UMP-saturated UPRTase (46 lM). s, GTP alone; e, CTP alone; ·, GTP binding in the presence of 50 lM CTP. (D) Binding of UMP to UMP-free UPRTase (4.9 lM) and the effects of other ligands on the binding interaction. s, UMP alone; e, UMP binding in the presence of 0.5 mM CTP; ·, UMP binding in the presence of GTP; n, UMP binding in the presence of 1 mM GTP and 1 mM PRPP. (E) Binding behavior of [14C]uracil to 4.9 lM of UMP-free UPRTase. d, uracil alone; e, in the presence of 100 lM PPi; n, in the pres- ence of 0.5 mM CTP; s, in the presence of 1 mM GTP. (F) Binding of 32PPi. d, PPi alone; e, in the presence of 63 lM uracil; no binding at all was detected in the pres- ence of 1 mM GTP (no symbols). The bind- ing parameters are summarized in Table 1.

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1445

CTP binds avidly to both the UMP-free UPRTase (Fig. 6B) and to the UMP-saturated enzyme (Fig. 6C) with a KD (cid:1) 5 lm, and it strongly inhibits binding of the activator GTP (Fig. 6C). The binding of the prod- uct UMP is very strong with KD (cid:1) 0.5 lm. The bind- ing may be increased slightly by the presence of CTP Ligand binding experiments were carried out both with the UMP-free preparation and the UMP saturated enzyme. A number of binding isotherms are shown in Fig. 6, and the parameters relating to these are sum- marized in Table 1. Figure 6A shows that the allosteric activator GTP (1 mm) reduces the concentration of PRPP needed for half saturation from 39 to 19 lm, and that CTP (50 lm) strongly inhibits the binding of PRPP and causes the binding curve to appear highly

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

Table 1. Ligand binding parameters. A negative sign preceding the enzyme concentration denotes that denatured and refolded UMP-free enzyme was used. A positive sign preceding the enzyme concentration denotes that UMP-saturated UPRTase was used.

Experiment

Ligand

Additions

N

Enzyme lM

L0.5 (lM)

nH

1 2 3 4 5 6 7 8 9 10

PRPP PRPP PRPP GTP GTP GTP GTP GTPa CTP CTPa

)26 )26 )26 )19 )19 +46 +46 +38 )19 )19

– 1 mM GTP 50 lM CTP – 1 mM PRPP – 1 mM PRPP 50 lM CTP – 1 mM PRPP

39 ± 10 18.4 ± 0.7 104 ± 86 35.9 ± 0.2 17.9 ± 0.9 85 ± 5 39 ± 2 207 ± 62 3.0 ± 0.3 290 ± 120

1.5 ± 0.4 1.7 ± 0.1 4 ± 3 4.0 ± 0.1 2.5 ± 0.5 2.9 ± 0.3 3.0 ± 0.3 3.0 ± 1.3 2.0 ± 0.5 1.1 ± 0.1

11 12 13 14 15

CTP UMPb UMPb UMPb UMPb

+42 )4.9 )4.9 )4.9 )4.9

0.26 0.30 0.33 0.39 0.39 0.47 0.57 0.17 0.44 Assuming N ¼ 0.5 0.68 0.50 0.47 0.44 0.20

5.8 ± 0.4 0.53 ± 0.10 0.53 ± 0.09 0.72 ± 0.10 7.5 ± 1.0

1.6 ± 0.2 1.1 ± 0.2 ¼ 1c ¼ 1c ¼ 1c

– – 0.5 mM CTP 1 mM GTP 1 mM GTP 1 mM PRPP c

1 mM GTP d

16 17 18 19

)4.9 )4.9 10 10

1 mM GTP

24 ± 42 19 ± 15 50 ± 21 No binding

0.47 ± 0.08 ¼ 1c ¼ 1c No binding

0.55 0.16 0.24 ~0

Uracild Uracil PPi e PPi

a The binding isotherms never approached saturation and the kinetic patameters, including the binding stoichiometry N, are thus very uncer- tain. b Similar parameters were seen with enzyme concentrations close to 20 lM. c The indication: nH ¼ 1 means that the Hill-coefficient was fixed to being ¼ 1 or in other words that the hyperbolic binding equation was used. d The L0.5 and N were determined from all binding data with uracil alone or in the presence of PPi or CTP. e The parameters given represent a fit to the data of PPi binding alone and the data of PPi binding including 63 lM uracil.

(0.5 mm) and inhibited weakly by GTP (1 mm), but clearly the combination of GTP (1 mm) and PRPP (1 mm) strongly inhibits UMP binding (Fig. 6D).

subunits) never approached one, but was close to 0.5. This binding stoichiometry has been remarkably repro- ducible and observed with several preparations of the enzyme, both the UMP-containing enzyme and the denatured and refolded enzyme used at different con- centrations in the binding assays.

Oligomeric state of UPRTase It is worth noticing that binding of the activator GTP and the substrate PRPP is a highly cooperative process, especially in presence of the inhibitor CTP when the Hill coefficient nH is close to four, while binding of UMP and the inhibitor CTP proceeds in a much less cooperative way.

in 50 mm succinate ⁄ phosphate

The binding behavior of [14C]uracil (Fig. 6E) was unusual. It appeared to exhibit negative cooperativity (nH ¼ 0.5) during binding to the ligand-free enzyme. Moreover, the binding of uracil was unaffected by the presence of PPi (100 lm) and CTP (0.5 mm), but it was considerably weakened by the presence of GTP (1 mm). Likewise the binding of [32P]PPi (Fig. 6F) was intriguing. The KD value for PPi was approximately 50 lm and was not significantly affected by the pres- ence of uracil, but when 1 mm GTP was added to the mixtures no binding of PPi could be detected in the tested concentration range ([PPi] ¼ 100 lm).

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1446

The effect of ligands on the size of the UPRTase was investigated by analyzing the sedimentation behavior of the UMP-free enzyme in 5–20% sucrose gradients, buffer, prepared pH 5.5, and containing 10 mm MgCl2, as described previously [4,20]. Three gradients were made: one with- out any ligands in the buffer; a second containing 0.5 mm CTP plus 0.5 mm UMP in addition to the buf- fer; and the third containing 1 mm PRPP and 1 mm GTP. Addition of the ligands did not change the sedi- mentation rate of the enzyme or the profile of the UPRTase activity peaks. The sedimentation constant, S20,w, was in the interval 5.6 ± 0.1 s, and the relative molecular mass calculated according to Martin and [20] was 98 000 ± 200, 95 800 ± 2200 and Ames In experiments where saturation was achieved, the binding stoichiometry (N ¼ maximal concentration of bound ligand divided by the concentration of enzyme

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

of substrates, products or allosteric effectors. Addition- ally, this enzyme does not exhibit the characteristic acceleration in the early phase of reactions started by the addition of PRPP and GTP, which reflects the assembly of (inactive) dimers to (active) tetramers for other enzymes [4]. The 96 500 ± 200 in the absence of ligands, in the presence of CTP and UMP, and in the presence of PRPP and GTP, respectively. Thus, the protein behaved as a tetramer under all tested conditions and a tetrameric structure was also seen in crystal structures of the S. solfataricus UPRTase in complexes with UMP and ⁄ or UMP plus CTP [17].

Discussion

Allosteric regulation

inhibition by CTP is unprecedented for UPRTases and not seen for other pyrimidine salvage inhibition is complex as it enzymes. The mode of depends on the concentration of UMP in the assays. It is clear that CTP by itself does not inhibit the UPR- Tase reaction strongly at high concentrations of PRPP and GTP, and that quite high concentrations of UMP are also required to significantly inhibit the enzyme under these conditions when the two ligands (CTP or UMP) are present individually. However, the enzyme becomes sensitive to inhibition by only very small amounts of UMP when CTP is also present. In the presence of 0.5 mm CTP (a concentration similar to the intracellular CTP pool under steady state growth conditions for most bacteria), the synthesis of UMP stopped completely when a few micromoles of UMP had accumulated in the assay mixtures. This kinetic behavior is difficult to treat in mathematical terms, but clearly the activation by GTP competes with the inhi- bition by CTP, because the inhibitory effect of CTP on enzyme activity is much stronger at 0.2 mm GTP than it is at a concentration of 1 mm GTP. The back- ward reaction, i.e. the pyrophosphorolysis of UMP, is regulated similarly to the UMP synthesis reaction by CTP and GTP.

The most interesting and unique feature of the uracil phosphoribosyltransferase from Sulfolobus solfataricus is the very strong stimulation seen with GTP and the powerful inhibition by CTP in the presence of UMP. In physiological terms such a regulation makes sense, as the need for pyrimidine nucleotide biosynthesis is expected to be high when the supply of purine nucleo- tides is high, and low when the end-product of the lin- ear pathway for pyrimidine nucleotide biosynthesis (CTP) has accumulated to high levels in the cells. An interplay between purine nucleotides (GTP, ATP) and pyrimidine nucleotides (CTP, UTP) is typical for regu- lation of pyrimidine nucleotide biosynthesis in many organisms, applying both to the regulation of pyr gene expression and enzyme activity [21]. However, the nucleotides usually regulate the activity of the first enzymes of the pyrimidine de novo pathway, the most well-known example being aspartate carbamoyltrans- ferase (ATCase), which is activated by ATP and inhib- ited in a synergistic fashion by CTP and UTP [22,23], while the salvage enzymes are usually unregulated.

The ligand binding studies showed that binding of CTP is incompatible with the binding of GTP and PRPP, because the binding isotherms for both of these ligands became highly sigmoidal in the presence of CTP, the Hill coefficient (nH) being near four. The presence of UMP also impaired binding of GTP and PRPP. In reciprocal experiments it was observed that the presence of PRPP and ⁄ or GTP increased the disso- ciation constants for the enzyme’s interaction with CTP and UMP, but the binding curves for these inhibi- tors remained (almost) hyperbolic, even when PRPP and GTP were included in the mixtures.

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1447

A stimulation of UPRTase activity by GTP was observed previously for the enzymes from E. coli [2–4] and T. gondii [5]. For these enzymes, GTP lowers the concentration of PRPP needed to give half maximal velocity without affecting the reaction rate (kcat) at sat- urating concentrations of PRPP. The activation mech- anism involves oligomerization of a dimeric form of each of the enzymes to a tetrameric form that is more active than the dimer. Both PRPP (the substrate) and GTP (the allosteric activator) stabilize the tetrameric form of the enzymes, which explains why GTP has no effect on the catalytic velocity at saturating concentra- tions of PRPP. In contrast, for the UPRTase from S. solfataricus, GTP causes a strong increase ((cid:1) 20- fold) of the catalytic rate (kcat) at saturating concentra- tions of PRPP, while the Km for PRPP is slightly elevated and the Km for uracil increased in parallel with kcat. Furthermore, it is clear that neither PRPP nor GTP influence the oligomeric state of this enzyme, as it sediments as a tetramer regardless of the addition The kinetic data and the binding behavior indicate that the tetrameric UPRTase from S. solfataricus exists in two conformations, namely an inactive T-state and an active R-state, according to Monod et al. [24]. PRPP and the allosteric activator GTP cooperate to stabilize the R-state, while the product UMP and the allosteric inhibitor CTP cooperate to stabilize the T-state. The strong sigmoidal nature of the binding curve for GTP (and PRPP) indicates that the T-state predominates the ligand-free form of the enzyme and

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

that the enzyme, saturated with UMP and CTP exclu- sively, exists in the T-form. Crystal structures repre- senting the inhibited form (the T-state) of the enzyme in complex with either UMP, or UMP and CTP, have recently been determined [17], but it has not yet been possible to obtain diffracting crystals of the enzyme in the active state (the R-form) in complex with PRPP and ⁄ or GTP.

Reaction mechanism

from S. solfataricus

The activity of UPRTase is remarkable in several ways. First, the very low pH optimum (5.0–5.6), shared by the enzyme from S. shi- batae [7] is unusual, as other UPRTases [3,25–28] and the phosphoribosyltransferases in general have optima around pH 8 [29], and because PRPP is not expected to form stable complexes with Mg2+ at this low pH [30,31]. The asymmetric shape of the pH activity pro- file indicates that more parameters influencing the activity (e.g. kcat, Km, GTP binding and concentrations of Mg2+ complexes with PRPP and ⁄ or GTP) are affected by pH in the pH interval 3–8. For this reason we did not investigate the pH activity profile further, and will not attempt an interpretetation of it in terms of kinetic constants.

The kinetics of a ping-pong reaction (i.e. parallel lines in double reciprocal plots of initial velocities vs. substrate concentration) was also observed for the PyrR protein (a regulatory RNA binding protein that also is a UPRTase) by Grabner and Switzer [34]. These authors attributed the kinetic behavior to an essenti- ally irreversible conformational change (E-PRPP to E¢-PRPP) of that enzyme in response to the binding of PRPP and preceding the binding of uracil. In this con- text, ‘an essentially irreversible conformational change’ means that the backwards change of E¢-PRPP to E-PRPP is much slower than all other kinetic steps in the reaction path. A similar conformational change in response to the binding of PRPP may explain the kin- etic behavior of UPRTase from S. solfataricus in the presence of GTP, but there may be other explanations. The recently determined crystal structures showed that UPRTase from S. solfataricus is composed of a tetr- amer of two asymmetric dimers [17]. When the enzyme was crystallized in the presence of an excess of UMP, this molecule was bound differently to the two active sites of each of the asymmetric dimers. In one site the conformation of UMP was constrained by numerous interactions with the enzyme, while the UMP molecule in the other site had a relaxed conformation with fewer to the enzyme. Additionally, one structure bonds formed in the absence of added UMP showed an enzyme with one site of each dimer being saturated with UMP while the other site was empty [17]. It is therefore the possibility that tempting to speculate about UPRTase from S. solfataricus works by a half-of-the- sites mechanism in which the binding of PRPP (assisted by GTP) to two sites per tetramer by inducing a con- formational change promoting the release of UMP from the two other sites to allow binding of uracil and a new catalytic cycle of the enzyme. Such a mechanism is in agreement with the ligand binding results, which indicate that PRPP binds first to the GTP-activated enzyme and that UMP is released as the final product. It is also in agreement with the apparent half-of-the- sites saturation we have observed with respect to sub- strates and products in these experiments, and with the observation by others that release of the nucleotide product often limits the reaction rate of the phospho- ribosyltransferases [33,35]. This is probably due to slow movements of loops covering the active sites [36].

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1448

The inability of the enzyme to catalyze exchange reactions implies that the protein works by a sequen- tial bi-bi kinetic mechanism, so that no chemical reac- tion occurs before both substrates are bound. Such a mechanism is consistent with the observed pattern of intersecting lines in double reciprocal plots of initial velocities vs. substrate concentration, when enzyme activity was assayed in the absence of GTP. It is also in line with the trend that phosphoribosyltransferases in general obey sequential mechanisms [32,33]. The lig- and binding studies suggest a random mechanism for the unactivated enzyme with no defined order of sub- strate binding and product release. However, the pres- ence of GTP alters this picture profoundly. Aside from increasing the turnover number (kcat) of the enzyme and strongly inhibiting the binding of uracil and PPi to the enzyme, the saturation with GTP made the enzyme obey the kinetics of a ping-pong reaction. This sug- gests that an irreversible kinetic step separates the binding of the two substrates (PRPP and uracil) to the GTP-activated enzyme. In a classical ping-pong mecha- nism this irreversible step is the release of the product that is formed in the first half reaction. However, as UPRTase does not catalyze any exchange reactions, it does not follow the reaction path of a classical ping- pong mechanism, with the overall reaction being com- posed of two half reactions. There is an apparent disagreement between our inability to saturate more than half of the sites in our ligand binding assays with the finding of crystal struc- tures in which all subunits of the tetramer contained CTP or UMP [17]. This conflict may be related to the fact that the ligand concentrations used to form the crystals were more than an order of magnitude higher

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

than the concentrations of ligands used for the binding assays presented here.

Conclusions

Technology A ⁄ S (Aarhus, Denmark). Poly(ethyleneimine)- impregnated cellulose thin-layer plates were made according to the procedure of Randerath and Randerath [38]. Dye- matrex Gel Green A was from Millipore Corporation (Bill- erica, MA, USA), Sephacryl S-200 and prepacked 10 mL Sephadex G25 columns (PD10) were from Pharmacia Bio- tech, diethylaminoethyl cellulose DE52 (preswollen) was from Whatman International Ltd (Maidstone, Kent, UK). chemicals Guanidinium hydrochloride and other fine were from Sigma, Serva (Heidelberg, Germany), Merck (Darmstadt, Germany), Calbiochem (EMD Biosciences Inc., Darmstadt, Germany) or Baker (Deventer, the Nether- lands). The bacterial strain NF1830, E.coli K-12 (recA1 ⁄ F’lacIq1 lacZ::Tn5) has been described previously [39].

Construction of an expression vectors

We have shown that GTP causes a conformational change in UPRTase, which dramatically increases the turnover number (kcat) of the enzyme without affecting the oligomeric state, and that CTP in combination with UMP almost completely inactivates the enzyme. The activation by GTP may be the result of an increase in the rate of on-enzyme chemistry or, perhaps more likely, an increase in the rate of UMP release from the enzyme, which may require movement of loops in the protein. Likewise, it is possible that the allosteric inhibitor CTP tightens the enzyme structure and slows down release of UMP. The data currently available do not permit an unambigous interpretation of the kinetic behavior of the enzyme, but we hope that future studies of the presteady state part of the reaction, in combination with crystal structures of the activated enzyme in complex with GTP and ⁄ or PRPP, will lead to a full understanding of the allosteric mechanism of this interesting enzyme.

Experimental procedures

radiochemicals

supplemented with

plates

agar

The upp gene was amplified by standard PCR techniques with VentR(cid:2) DNA polymerase using chromosomal DNA of the S. solfataricus strain P2. The upstream primer, Sup1, 5¢-CACAGAATTCAGGAGAGAAATAATGCCATTATAC GTAATCGATAAACC-3¢ contained a ribosome binding site and a cleavage site for EcoRI, while the downstream pri- mer, Sup2, 5¢-CGGGATCCGAATCATCACCCAAATGCT CTATCTCCA-3¢ contained the stop-codon and a BamHI site. Those parts of the primers corresponding to S. solfa- taricus DNA sequence are shown in bold. The PCR frag- ment was digested with EcoRI and BamHI and ligated into plasmid pUHE23-2 [18], previously digested with the same two endonucleases and treated with alkaline phosphatase to prevent self-ligation. The ligated mixture was transformed into host E. coli strain NF1830, which carries an F ¢lacI q1 episome and overproduces the lac-repressor so that tran- scription of the cloned gene is repressed until induction by isopropyl thio-b-d-galactoside [39], and plated on Luria– ampicillin Bertani (100 lgÆmL)1). The selected plasmid (pLSF2) contained the upp gene and the sequence was found to be identical to the sequence of the published sequence of the upp gene of S. solfataricus, the open reading frame SSO0231 [16].

Materials

The nucleoside triphosphates were Ultrapure(cid:2) NTPs from Pharmacia Biotech (Uppsala, Sweden). Other nucleotides, uracil and PRPP were from Sigma (St Louis, MO, USA). [2–14C]uracil and [2–14C]uridine-5¢- The [32P]GTP[aP], monophosphate (UMP) were from Sigma, [32P]CTP[aP] and [32P]ATP[cP] were from Amersham Phar- macia Biotech AB (Uppsala, Sweden). [32P]Pyrophosphate was from NEN Life Science Products (Boston, MA, USA). [32P]PRPP was prepared from [32P]ATP[cP] and ribose-5- phosphate as described previously [4] using E. coli PRPP synthetase to catalyze the reaction. Restriction enzymes and T4-DNA ligase were from Promega (Madison, WI, USA), and VentR(cid:2) DNA polymerase was from New England Bio- labs (Beverly, MA, USA). The BigDye(cid:2) Terminator Cycle Sequencing v2.0 Ready Reaction kit with AmpliTaq(cid:2) po- lymerase for the ABI Prism(cid:2) sequencing apparatus were from PE Biosystems (Foster City, CA, USA). Purified chro- mosomal DNA of the S. solfataricus strain P2 was a gift from Q. She (Institute of Molecular Biology, University of Copenhagen, Denmark). Yeast alcohol dehydrogenase and bovine liver catalase were from Boehringer (Mannheim, Germany). E. coli orotate phosphoribosyltransferase was prepared as described previously [37] and E. coli PRPP synthetase was a gift from M. Willemoes (Center for Crys- tallographic Studies, University of Copenhagen, Denmark). Synthetic DNA oligonucleotides were bought from DNA

A two litre LB broth culture of the E.coli strain NF1830 transformed with pLSF2 was grown with vigorous aeration at 37 (cid:1)C. Isopropyl thio-b-d-galactoside (0.5 mm) was added to the culture at D436 ¼ 0.5 to induce expression of the upp gene, and growth was continued into stationary phase over- night. Cells (9 g wet weight) were harvested by centrifuga- tion in a Sorvall G53 rotor at 5000 r.p.m. for 15 min at 4 (cid:1)C, resuspended in 200 mL of 0.1 m Tris ⁄ HCl, pH 7.3, 2 mm EDTA, and disrupted by ultrasonic treatment. The extract was cleared by centrifugation at 10 000 r.p.m. in a Sorvall SS34 rotor for 15 min at 4 (cid:1)C giving a crude extract (54 mL, Fraction 1). Following the addition of UMP to a

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1449

Cell growth and enzyme purification

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

Preparation of UMP-free enzyme was accomplished by gel filtration under denaturing conditions. The protein solution (625 lL, (cid:1) 6 mg) in TE buffer was mixed with 8 m guanidi- nium chloride (GdHCl; 1875 lm also in TE buffer) to give a final concentration of guanidinium chloride of 6 m. The solution was left at room temperature ((cid:1) 25 (cid:1)C) for 30 min. The sample (2.5 mL) was then loaded onto a prepacked 10 mL column of Sephadex(cid:3) G-25M (PD-10 column from Amersham Pharmacia equilibrated with 6 m GdHCl in TE buffer. The liquid appearing from the column was discar- ded. Subsequently, 3.5 mL of 6 m GdHCl in TE buffer was added to the column and the eluted fraction containing the macromolecular substances (UPRTase) was collected. The application of an additional 3.5 mL of 6 m GdHCl in TE buffer resulted in elution of the small molecules and this fraction was also collected. The absorption spectra (range 240–310 nm) of all three fractions were recorded using 6 m GdHCl in TE buffer as a reference standard. Figure 2B shows the spectra and they reveal that this procedure quan- titatively separated the enzyme from the bound ligand. The unfolded UMP-free enzyme (3.5 mL) in 6 m GdHCl in TE buffer was dialyzed with stirring first against 0.25 L TE buf- fer for 1 h at room temperature and then against 0.75 L TE buffer at 6 (cid:1)C overnight. Some protein precipitated in the dialysis bag and was removed by centrifugation (SS34, 5000 r.p.m., 15 min, 4 (cid:1)C). About two thirds of the protein refolded and regained activity. This UMP-free preparation of UPRTase was used within four days of storage at 4 (cid:1)C.

Preparation of UMP-free enzyme

to 12 UÆmg)1

final concentration of 2 mm the extract was heated and left at 75 (cid:1)C for 15 min. Denatured protein was removed by centrifugation (SS34, 15 000 rpm, 30 min, 4 (cid:1)C). The pro- tein in the supernatant (Fraction 2, 46 mL) was precipitated by the addition of solid ammonium sulfate to give 65% saturation. The solution was stirred at 6 (cid:1)C for 30 min; the precipitated protein was collected by centrifugation (SS34, 11 000 r.p.m., 15 min, 4 (cid:1)C), dissolved in and dialyzed exhaustively against 10 mm Tris ⁄ HCl, pH 8 (Fraction 3, 24 mL). The fraction was pumped (1 mLÆmin)1) onto a col- umn of DEAE–cellulose (Whatman DE52, diameter 1.5 cm, hight 30 cm) equilibrated with 10 mm Tris ⁄ HCl, pH 8. Fol- lowing application the column was washed with 100 mL of 10 mm Tris ⁄ HCl, pH 8.0, 0.1 mm EDTA (TE buffer). Sub- sequently the enzyme was eluted with a 200 mL linear gradi- ent from 0 to 200 mm NaCl in 10 mm Tris ⁄ HCl, pH 8, followed by 100 mL of 200 mm NaCl in 10 mm Tris ⁄ HCl, pH 8, and 5 mL fractions were collected. The fractions con- taining most UPRTase, eluting with a peak at 85 mm NaCl, were pooled (Fraction 4, 30 mL) and applied directly to a 20 mL column of Dyematrex Gel Green A. The column was washed with 50 mL 10 mm Tris ⁄ HCl, pH 8, and the enzyme was eluted with a 100 mL linear gradient from 0 to 1 m NaCl in 10 mm Tris ⁄ HCl, pH 8 (Fraction 5). It eluted from the column with a peak at 0.26 m NaCl. The active fractions were pooled and concentrated to (cid:1) 5 mL in a Centriprep(cid:2) spin device (Amicon, Bedford, MA, USA) and passed through a 2.5 · 80 cm Sephacryl S200 gel-filtration column. The peak fractions were pooled, concentrated to approxi- mately 10 mgÆmL)1 and dialyzed against 10 mm Tris ⁄ HCl, pH 8, 0.1 mm EDTA, containing 10% (v ⁄ v) glycerol. The gel-filtration step did not give a measurable increase of the specific activity, but was carried out to remove small peptides potentially present in the preparation. During the purification the specific activity of the protein prepar- ation increased from 0.4 UÆmL)1 (about 30-fold). The total yield measured in terms of activity was 40% and the loss in each individual step did not exceed 20%. The enzyme was either stored frozen at )20 (cid:1)C (for crystallographic trials) or in a nonfrozen at )20 (cid:1)C after 10 mm condition against dialysis Tris ⁄ HCl, pH 8.0, containing 0.1 mm EDTA and 50% (v ⁄ v) glycerol, for kinetic analysis.

Assays of enzyme activity

The standard assay of enzyme activity was carried out at 60 (cid:1)C and contained in a volume of 50 lL of 50 mm succi- nate [sodium phosphate pH 5.5, 10 mm MgCl2, 0.6 mm PRPP, 1 mm GTP, enzyme ((cid:1) 15 ng UPRTase), and ((cid:1) 0.2 TBqÆmol)1)]. The mixtures, 100 lm [2–14C]uracil minus uracil, were assembled at room temperature and pre- warmed at 60 (cid:1)C. Reactions were started at time t ¼ 0 by the addition of 10 lL of [14C]uracil. Samples (10 lL) were withdrawn after 2, 5 and 10 min, and applied to PEI– cellulose thin-layer plates, which stopped the reaction. Ura- cil was separated from UMP by chromatography in water on 12 cm long PEI thin-layer plates. The plates were dried and the radioactivity in the substrate and product was determined by counting in an instant imager (Canberra Packard, Merriden, CT, USA). One unit of enzyme activity is defined as the amount of UPRTase that converts 1 lmol uracil to UMP under these conditions.

To analyze the content of UMP in UPRTase, 200 lL enzyme solution (1 mgÆmL)1, dialyzed against TE buffer) was mixed with 20 lL of a 1 : 1 mixture of concentrated sulfuric acid and perchloric acid (70%). After incubation at 4 (cid:1)C for 30 min, the precipitated protein was removed by centrifugation at 20 000 g for 10 min at 4 (cid:1)C and the UV absorption spectrum of the acid supernatant was recorded. A sample of the buffer (the dialysate) was treated similarly and used as a standard.

Determination of optimal pH was made using similar reactions but at different pH. In the interval 2.6 ¼ pH ¼ 7.1, buffers were made by mixing 0.2 m succinic acid with 0.2 m Na2HPO4 to the required pH. In the interval 6.9 ¼ pH ¼ 7.9, the buffer was made by titrating a 0.2 m Tris

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1450

Analysis of UMP content

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

solution to the desired pH by adding concentrated hydro- chloric acid. The actual pH values were measured at 60 (cid:1)C in buffers diluted four times (50 mm).

or the equation for a two substrate ping-pong mechanism:

v ¼ Vmax½A(cid:2)½B(cid:2)=ð½A(cid:2)½B(cid:2) þ Km;A½B(cid:2) þ Km;B½A(cid:2) þ Kia Km;BÞ ð3Þ

The ligand binding data were fitted either to the equation for hyperbolic saturation

ð4Þ v ¼ Vmax½A(cid:2)½B(cid:2)=ð½A(cid:2)½B(cid:2) þ KM;A½B(cid:2) þ KM;B½A(cid:2)Þ Ligand binding assays

or the Hill equation

ð5Þ ½Lb(cid:2) ¼ Bmax½Lf(cid:2)=ðKD þ ½Lf(cid:2)Þ

0:5 þ ½Lf(cid:2)nH Þ

[E]t

The radioactively labeled ligands (0.2–2 TBqÆmol)1) were incubated with 8–50 lm UPRTase, together with activators or inhibitors at the indicated concentrations, in a volume of 200 lL in the presence of 50 mm succinate (sodium phos- phate pH 5.5 and 10 mm MgCl2). After assembly at room temperature the mixtures were incubated at 60 (cid:1)C for 20 min and cooled again to room temperature. Samples (150 lL) were applied to Ultrafree-MC centrifugation filter devices (Amicon) and centrifuged at 5000 g. Liquid passing through the membrane during the first few seconds was dis- carded, while the liquid that passed through the membrane during the next 2–5 min of centrifugation was collected to determine the concentration of free ligand. The total con- centration of the ligand (Lt) was determined by measuring the radioactivity in 20 lL samples of the mixtures with- drawn prior to the centrifugation step, and the concentra- tion of free ligand (Lf) by measuring the radioactivity in 20 lL of the liquid that had passed through the filter.

where v is the initial reaction velocity, Vmax ¼ kcat[E]t is the maximal velocity at a given enzyme concentration, is the total enzyme concentration given as monomers, kcat is the turnover number, [S] is the concentration of substrate when only one substrate is varied, [A] is the concentration of one substrate (the first to bind), [B] the concentration of the other substrate, Km,A is the Km for substrate A, Km,B is the Km for substrate B, S0.5 is the concentration of substrate that gives half maximal reaction velocity, [Lb] is the concentration of bound ligand and [Lf] the concentration of free ligand, L0.5 is the concentration of ligand giving half maximal saturation, Bmax is the maximal concentration of bound ligand at a given concentration of enzyme, and nH is the Hill coefficient.

ð6Þ ½Lb(cid:2) ¼ Bmax½Lf(cid:2)nH =ðLnH

Size determination by ultracentrifugation

Acknowledgements

for

the UPRTase

The size of native UPRTase was analyzed by sedimenta- tion through 5–20% sucrose gradients at 5 (cid:1)C using a Beckman SW41 swinging bucket rotor at 39 000 r.p.m. for 25 h at 5 (cid:1)C as described by Martin and Ames [20] and from E. coli by used previously Jensen and Mygind [4]. Bovine liver catalase, yeast alcohol dehydrogenase and E. coli orotate phosphoribosyltrans- ferase were included as markers. The sucrose gradients were made up in 50 mm succinate (sodium phosphate pH 5.5 and 10 mm MgCl2). One gradient was made with- out ligands, while another gradient contained GTP and PRPP (1 mm each), and a third contained CTP and UMP (0.5 mm of each ligand).

We want to thank Dr Martin Willemoes for the supply of PRPP synthase, and Dr Qunxin She for making sequences of the S. solfataricus genome available to us prior to publication and for the supply of chromoso- mal S. solfataricus DNA. We also acknowledge the financial support from The Danish National Science Research Council in the form of a grant to K.F.J., and the Ph.D. grant to S.A. from the Faculty of Science, University of Copenhagen.

References

1 Jensen KF (1983) Metabolism of 5-phosphoribosyl

The data from kinetic and ligand binding experiments were fitted to the equations below using the BIOSOFT program ultrafit(cid:2) for Macintosh.

Kinetic data were fitted to either the hyperbolic satura-

1-pyrophosphate (PRPP) in Escherichia coli and Salmo- nella typhimurium. In Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganisms (Munch- Petersen A, ed), pp. 1–25. Academic Press, London. 2 Fast R & Sko¨ ld O (1977) Biochemical mechanism for

tion equation:

ð1Þ

v ¼ Vmax½S(cid:2)=ðKm þ ½S(cid:2)Þ

uracil uptake regulation in Escherichia coli B. Allosteric effects on uracil phosphoribosyltransferase under strin- gent conditions. J Biol Chem 252, 7620–7624.

or the Hill equation

Treatment of kinetic and ligand binding data

0:5 þ ½S(cid:2)nH Þ

3 Rasmussen UB, Mygind B & Nygaard P (1986) Purifi- cation and some properties of uracil phosphoribosyl- transferase from Escherichia coli K12. Biochim Biophys Acta 881, 268–275.

Initial velocity data were fitted to the equation for a sequen- tial bi-bi mechanism:

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1451

ð2Þ v ¼ Vmax½S(cid:2)nH =ðSnH

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

4 Jensen KF & Mygind B (1996) Different oligimeric

states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli. Eur J Biochem 240, 637–645.

17 Arent S, Harris P, Jensen KF & Larsen S (2005) Allosteric regulation and communication between subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus. Biochemistry 44, 883–892.

5 Schumacher MA, Bashor CJ, Song MH, Otsu K, Zhu S, Parry RJ, Ullman B & Brennan RG (2002) The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase. Proc Natl Acad Sci USA 99, 78–83.

18 Deuschle U, Kammerer W, Gentz R & Bujard H (1986) Promoters of E. coli. A hierarchy of in vivo strength indicates alternate structures. EMBO J 5, 2987–2994. 19 Gill SC & von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182, 319–326.

6 Dai Y-P, Lee CS & O’Sullivan WJ (1995) Properties of uracil phosphoribosyltransferase from Giardia intesti- nalis. Int J Parasitol 25, 207–214.

7 Linde L & Jensen KF (1996) Uracil phosphoribosyl-

20 Martin RG & Ames BN (1961) A method for deter- mining the sedimentation behaviour of enzymes: application to protein mixtures. J Biol Chem 236, 1372– 1379.

21 Jensen KF (1989) Regulation of Salmonella typhimurium pyr gene expression: effect of changing both purine and pyrimidine nucleotide pools. J Gen Microbiol 135, 805– 815.

transferase from the extreme thermophilic archaebacter- ium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP. Biochim Bio- phys Acta 1296, 16–22.

8 Natalini P, Rugguirerri S, Santarelli I, Vita A & Magni G (1979) Baker’s yeast UMP: pyrophosphate phospho- ribosyltransferase. J Biol Chem 254, 1558–1563.

22 Wild JR, Loughrey-Chen SJ & Corder TS (1989) In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase. Proc Natl Acad Sci USA 86, 46–50.

23 England P & Herve´ G (1992) Synergistic inhibition of

Escherichia coli aspartate transcarbamylase by CTP and UTP: Binding studies using continous-flow dialysis. Bio- chemistry 31, 9725–9732.

9 Jensen HK, Mikkelsen N & Neuhard J (1997) Recombi- nant uracil phosphoribosyltransferase from the thermo- phile Bacillus caldolyticus: Expression, purification, and partial characterisation. Protein Expr Purif 10, 356–364. 10 Kadziola A, Neuhard J & Larsen S (2002) Structure of product-bound Bacillus caldolyticus uracil phosphoribo- syltransferase confirms ordeder sequential substrate binding. Acta Cryst D D58, 936–945.

24 Monod J, Wyman J & Changeux J-P (1965) On the nat- ure of allosteric transitions: a plausible model. J Mol Biol 12, 88–118.

11 Schumacher MA, Carter D, Scott DM, Roos DS, Ull-

25 Asai T, Lee CS, Chandler A & O’Sullivan WJ (1990)

Purification and characterization of uracil phosphoribo- syltransferase from Crithidia luciliae. Comp Biochem Physiol 95B, 159–163.

man B & Brennan RG (1998) Crystal structures of Tox- oplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and pro- drug binding. EMBO J 17, 3219–3232.

12 Joint Center for Structural Genomics. (2005) Crystal

26 Alloush HM & Kerridge D (1994) Characterisation of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogen Candida albicans. Myco- pathologia 125, 129–141.

structure of uracil phosphoribosyltransferase (TM0721) from Thermotoga maritima at 2.30 A˚ resolution. In press.

27 McIvor RS, Wohlhueter RM & Plagemann PGW

13 Scapin G, Grubmeyer C & Sacchettini JC (1994) Crystal structure of orotate phosphoribosyltransferase. Biochem- istry 33, 1287–1294.

(1983) Uracil phosphoribosyltransferase from Achole- plasma laidlawii: Partial purification and kinetic proper- ties. J Bacteriol 156, 192–197.

28 Plunkett W & Moner JG (1978) UMP Pyrophosphory- lase of Tetrahymena pyriformis. Arch Biochem Biophys 187, 264–271.

14 Eriksen TA, Kadziola A, Bentsen AK, Harlow KW & Larsen S (2000) Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthe- tase. Nat Struct Biol 7, 303–308.

15 Lundegaard C & Jensen KF (1999) Kinetic mechanism

29 Grubmeyer C & Wang G (1998) Making a nucleotide: structure and function of orotate phosphoribosyltrans- ferase. Paths Pyrimidines 6, 1–11.

30 O’Sullivan W & Smithers GW (1979) Stability constants

of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site. Biochemistry 38, 3327–3334.

for biologically important metal-ligand complexes. Methods Enzymol 63, 294–336.

31 Willemoe¨ s M & Hove-Jensen B (1997) Binding of divalent magnesium by Escherichia coli phospho- ribosyl diphosphate synthetase. Biochemistry 36, 5078–5083.

16 She Q, Singh RK, Confalonieri F, Zivanovic Y, Allard G, Awayez MJ, Chan-Weiher CC, Clausen IG, Curtis BA, De Moors A, et al. (2001) The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc Natl Acad Sci USA 98, 7835–7840.

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1452

K. F. Jensen et al.

Regulation of uracil phosphoribosyltransferase from S. solfataricus

36 Wang GP, Cahill SM, Xiaohong L, Girvin ME &

32 Bhatia MB, Vititsky A & Grubmeyer C (1990) Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium. Biochemistry 29, 10480–10487.

33 Xu Y, Eads J, Sacchettini JC & Grubmeyer C (1997) Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl trans- fer chemistry. Biochemistry 36, 3700–3712.

Grubmeyer C (1999) Motional dynamics of the catalytic loop in OMP synthase. Biochemistry 38, 284–295. 37 Poulsen P, Bonekamp F & Jensen KF (1984) Structure of the Escherichia coli pyrE operon and control of pyrE expression by a UTP modulated intercistronic attenua- tion. EMBO J 3, 1783–1790.

38 Randerath K & Randerath E (1967) Thin-layer separa-

tion methods for nucleic acid derivatives. Methods Enzy- mol 12A, 323–347.

39 Andersen JT, Poulsen P & Jensen KF (1992) Attenua-

34 Grabner GK & Switzer RL (2003) Kinetic studies of the uracil phosphoribosyltransferase reaction catalyzed by the Bacillus subtilis pyrimidine attenuation regulatory protein PyrR. J Biol Chem 278, 6921–6927.

tion in the rph-pyrE operon of Escherichia coli and pro- cessing of the dicistronic mRNA. Eur J Biochem 206, 381–390.

35 Wang G, Lundegaard C, Jensen KF & Grubmeyer C (1999) Kinetic mechanism of OMP synthase: a slow physical step following group transfer limits catalytic rate. Biochemistry 38, 275–283.

FEBS Journal 272 (2005) 1440–1453 ª 2005 FEBS

1453