Mechanistic Characterization of Toxoplasma gondii ThymidylateSynthase (TS-DHFR)-Dihydrofolate ReductaseEVIDENCE FOR A TS INTERMEDIATE AND TS HALF-SITES REACTIVITY*

Received for publication, July 1, 2002, and in revised form, August 15, 2002Published, JBC Papers in Press, August 20, 2002, DOI 10.1074/jbc.M206523200

Eric F. Johnson‡, Wolfgang Hinz‡, Chloe E. Atreya‡, Frank Maley§, and Karen S. Anderson‡¶

From the ‡Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut 06520 andthe §New York State Department of Health, Albany, New York 12201

This study describes the use of rapid transient kineticmethods to characterize the bifunctional thymidylatesynthase-dihydrofolate reductase (TS-DHFR) enzymefrom Toxoplasma gondii. In addition to elucidating thedetailed kinetic scheme for this enzyme, this work pro-vides the first direct kinetic evidence for the formationof a TS intermediate and for half-sites TS reactivity inhuman and Escherichia coli monofunctional TS and inT. gondii and Leishmania major bifunctional TS-DHFR.Comparison of the T. gondii TS-DHFR catalytic mecha-nism to that of the L. major enzyme reveals the mecha-nistic differences to be predominantly in DHFR activity.Specifically, TS ligand induced domain-domain communi-cation involving DHFR activation is observed only in theL. major enzyme and, whereas both DHFR activities in-volve a rate-limiting conformational change, the changeoccurs at different positions along the kinetic pathway.

Thymidylate synthase (TS)1 and dihydrofolate reductase(DHFR) are essential metabolic enzymes and established tar-gets for anti-cancer and anti-microbial drugs (1–3). Although,in most species, including humans, TS and DHFR activitiesreside on separate monofunctional enzymes, several protozoanparasites have these activities expressed on a single polypep-tide chain that comprises a bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR)2 enzyme (4–8). One ofthese protozoa, Toxoplasma gondii, is prevalent and problem-atic in the United States (8). Opportunistic toxoplasmosis isoften associated with the onset of the clinical AIDS syndrome

and is a primary cause of suffering and death in AIDS patients(6). Enzymes unique to the parasite, including the bifunctionalTS-DHFR, are optimal targets for the development of newanti-parasitic drugs.

As illustrated in Scheme 1, TS catalyzes the only de novosource of deoxythymidine monophosphate (dTMP) from de-oxyuridine monophosphate (dUMP). The reaction uses (6R)-L-5,10-methylenetetrahydrofolate (CH2H4F) as a cofactor for theone carbon transfer reaction generating 7,8-dihydrofolate(H2F) in the process. DHFR regenerates the fully reduced formof the folate (6R)-5,6,7,8-tetrahydrofolate (H4F) from H2F usingNADPH as a cofactor and generating NADP� in the process.H4F can then by primed for subsequent one-carbon transferreactions in the cell. In the presence of dUMP, NADPH, andCH2H4F, the bifunctional TS-DHFR enzymes catalyze the con-version of CH2H4F directly to H4F.

Scheme 2 illustrates the detailed catalytic mechanism thathas been proposed for the TS reaction (9). Following orderedsubstrate binding, in which dUMP binds first, a conformationalchange takes place whereby the C-terminal tetrapeptide of TScloses over the substrates to create an active site cavityshielded from solvent (step 1) (1, 10–14). It has been suggestedthat this is followed by the formation of an iminium ion involv-ing the bridge methylene and N-5 of CH2H4F (step 2) (15).

It is this highly reactive electrophilic iminium ion that hasbeen proposed to be the reactive form of the cofactor. In fact,the structure of a TS mutant lacking a C-terminal valine andcrystallized with CH2H4F and 5-fluorodeoxyuridine monophos-phate (FdUMP) in which (6R)-L-5-hydroxymethyltetrahydrofo-late (HO-CH2H4F) is bound at the active site provided struc-tural evidence supporting the formation of this iminium ionduring TS catalysis (10). Kinetic isotope effect studies suggestthat it is bound CH2H4F, potentially in its iminium ion form,that accumulates at the active site (9). Several steps in rapidequilibrium ensue leading up to an overall rate-limiting stepinvolving hydride transfer to form the products dTMP and H2F(steps 3–6) (9).

All known TS enzymes, with the exception of a class ofrecently discovered flavin-dependent tetrameric TS enzymesfrom several non-symbiotic microbes, exist as a TS homodimer(16). It has been suggested that TS is a half-sites-reactiveenzyme in which only one TS site of dimeric TS productivelybinds substrates (17–19). Until now, however, definitive kineticevidence was lacking and the issue of whether TS is actually ahalf-sites-reactive enzyme has remained unresolved (9).

There is structural and kinetic evidence to suggest thatbifunctional TS-DHFRs from certain protozoa, including Leish-mania major and T. gondii exhibit electrostatic substrate chan-neling in which H2F is directly transferred between the TS andDHFR active sites without release into bulk solvent (5, 20–22).

* This work was supported by National Institutes of Health (NIH)Grant AI44630 and American Cancer Society Grant RPG-98-027-01-CDD (to K. S. A.), NIH Grant CA44355 (to F. M.), and NIH MedicalScientist Training Program Grant GMO7205 (to C. E. A.). The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¶ To whom correspondence should be addressed: Dept. of Pharmacol-ogy, School of Medicine, Yale University, 333 Cedar St., New Haven, CT06520. Tel.: 203-785-4526; Fax: 203-785-7670; E-mail: karen.anderson@yale.edu.

1 The abbreviations used are: TS, thymidylate synthase; DHFR, di-hydrofolate reductase; TS-DHFR, bifunctional thymidylate synthase-dihydrofolate reductase; dTMP, deoxythymidine monophosphate;dUMP, deoxyuridine monophosphate; CH2H4F, (6R)-L-5,10-methylenetetrahydrofolate; H2F, 7,8-dihydrofolate; H4F, (6R)-5,6,7,8-tetrahydro-folate; FdUMP, 5-fluorodeoxyuridine monophosphate; HO-CH2H4F,(6R)-L-5-hydroxymethyltetrahydrofolate; CCD, charge-coupled device;HPLC, high-performance liquid chromatography; PDB, Protein DataBank; FRET, fluorescence resonance energy transfer.

2 The bifunctional enzyme is sometimes referred to as DHFR-TS,because DHFR comprises the N-terminal portion of the protein. Thisreport uses the TS-DHFR designation, because, mechanistically, the TSreaction precedes the DHFR reaction.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 45, Issue of November 8, pp. 43126–43136, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Previous kinetic studies from our group provided support forsubstrate channeling in TS-DHFR from L. major (23). Theserapid transient kinetic studies on L. major also provided directkinetic evidence for domain-domain communication in L. majorTS-DHFR in which ligation of the TS active site with FdUMPand CH2H4F to form the covalent FdUMP�CH2H4F�TS-DHFRcovalent complex resulted in activation of DHFR chemistryfrom 14 to 120 s�1 (23).

In this work, we set out to address fundamental mechanisticquestions about the TS-DHFR from T. gondii; a detailed un-derstanding of which might aid in novel anti-parasite drugdevelopment against this medically relevant organism. Specif-ically, we asked: What are the rate-limiting steps in the reac-tions catalyzed by this bifunctional enzyme? Can we gain anynew insight into the catalytic mechanism of TS from this bi-functional enzyme? How does this bifunctional TS-DHFR com-pare with the previously characterized TS-DHFR from L. majorfor which a structure is known?

In this report, we describe a transient kinetic analysis usingrapid chemical quench and stopped-flow methods to providethe first in-depth characterization of the reaction pathway forthe bifunctional TS-DHFR from T. gondii. In addition to eluci-dating the detailed mechanism for the T. gondii TS-DHFRenzyme, this study provides the first direct kinetic evidence forthe formation of a TS intermediate and for half-sites TS reac-tivity. Subsequent analysis of L. major TS-DHFR, Escherichiacoli TS, and human TS provided evidence for an intermediateand for half-sites TS reactivity in these enzymes as well.

MATERIALS AND METHODS

Enzymes—A clone harboring the p02CLSA-4 plasmid expressed inan E. coli Rue 10 expression vector was used following previouslydescribed methods to obtain bifunctional L. major TS-DHFR of highpurity (23–25). The TS-DHFR was further purified using a AmershamBiosciences Superdex� 75 HiLoad� (26/60) gel filtration column to rid ofresidual H2F contaminant. T. gondii TS-DHFR was similarly preparedfrom E. coli BL21-DE3 cells (Stratagene) freshly transformed withPET15b plasmid containing the coding sequence for T. gondii TS-DHFR. The T. gondii TS-DHFR plasmid was a generous gift from Dr.David S. Roos. Enzyme concentrations were determined spectrophoto-metrically at 280 nm using a molar extinction coefficient of 67,800M�1cm�1 for L. major TS-DHFR, 78,800 M�1cm�1 for T. gondii TS-DHFR,43,800 M�1cm�1 for human TS, and 52,150 M�1cm�1 for E. coli TS.

Chemicals—All buffers and other reagents employed were of thehighest commercial purity. Millipore ultrapure water was used for allsolutions. 7,8-Dihydrofolate (H2F) was chemically prepared by the re-duction of folate with sodium hydrosulfite (26). (6R,S)-5,6,7,8-Tetrahy-SCHEME 1

SCHEME 2

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drofolate (H4F) was obtained from Schirks Laboratories, Switzerland.Radiolabeled H2F was synthesized by sodium hydrosulfite reduction oftritium-labeled [3�,5�,7,9-3H]folic acid obtained from Moravek Bio-chemicals (Brea, CA). Radiolabeled and unlabeled CH2H4F were pre-pared by enzymatic conversion of radiolabeled and unlabeled H2F,respectively, to form (6R,S)-5,6,7,8-tetrahydrofolate (H4F) and subse-quent condensation with formaldehyde (27). Both H2F and CH2H4Fwere purified by DE-52 (Whatman Co.) anion exchange chromatogra-phy as previously described (28). 14C-Labeled dUMP was obtained fromMoravek Biochemicals (Brea, CA). The concentrations of H2F (�282 �28,000 M�1cm�1), H4F (�297 � 28,000 M�1cm�1), and CH2H4F (�290 �32,000 M�1cm�1) were determined spectrophotometrically (29, 30).NADPH (�340 � 6,220 M�1cm�1), NADP� (�260 � 18,000 M�1cm�1),dUMP (�260 � 10000 M�1cm�1), and dTMP (�260 � 8,400 M�1cm�1) werepurchased from Sigma, and their concentrations were determined usingreported molar extinction coefficients. Experiments were carried out at25 °C in 50 mM Tris buffer (pH 7.8) containing 1 mM EDTA, 25 mM

MgCl2, and 10 mM dithiothreitol. Buffer solutions were purged withargon prior to use.

Stopped-flow Measurements—Stopped-flow measurements were per-formed using a Kintek SF-2001 apparatus (Kintek Instruments, Aus-tin, TX) as previously described (23). The data were collected over agiven time interval using a PC and software provided by Kintek Instru-ments. In experiments designed to measure dissociation rate constants,the trapping ligand was used at a concentration of �5-fold excess overthat of the bound ligand to allow analysis as a pseudo-first order rateconstant. Coenzyme FRET was utilized in DHFR experiments and in allligand association and dissociation experiments involving NADPH. Forthese experiments, a monochromator was set to 287 nm on the inputand changes in NADPH FRET were monitored with an output filter at450 nm. For TS and TS-DHFR experiments, changes in absorbance at340 nm were monitored. For all other ligand binding experiments,changes in fluorescence with excitation at 287 and emission at 340 nmwere monitored. Due to ordered binding to TS in which nucleotide bindsfirst, followed by folate; H2F binding to the DHFR domain could beisolated from that to the TS domain by performing the experiments inthe absence of nucleotide (dUMP or dTMP). Likewise, binding of H2F toTS could be separately assessed by preincubating enzyme with aDHFR-saturating concentration of H2F (�5 �M) prior to mixing with alarge excess of nucleotide and an equimolar concentration of H2F. Thecombination of rapid chemical quench and stopped-flow methods al-lowed for an accurate interpretation of fluorescence and absorbancesignals.

CCD-array Stopped-flow Measurements—CCD-array stopped-flowmeasurements were performed using a Kintek SF-2001 apparatus (Kin-tek Instruments, Austin, TX) and detected with an Ocean OpticsPC2000 CCD linear silicon array detector. Absorption measurementswere taken for 2.1 s (integration time of 3 ms) from 220 to 450 nm (over649 elements), and the data were analyzed using a PC and Specfit/32TM

Software (Spectrum Software Associates).Rapid Chemical Quench—Rapid chemical quench experiments were

performed using a Kintek RFQ-3 Rapid Chemical Quench apparatus(Kintek Instruments, Austin, TX). The reactions were initiated by mix-ing enzyme solution (15 �l) with radiolabeled substrate (15 �l, �20,000dpm). In all cases, the concentrations of enzyme and substrates cited inthe text are those after mixing and during the reaction. Reactionsutilizing radiolabeled folates were terminated by quenching with 67 �lof 0.78 N KOH, 10% sodium ascorbate, and 200 mM 2-mercaptoethanol.Ascorbate and 2-mercaptoethanol were added to prevent oxidative deg-radation of H4F after quenching and resulted in a pH of 12.6 for thebase quench solution. Because CH2H4F is more stable under basicconditions, its solutions were maintained at a basic pH (9.5) untilmixing with enzyme solution, providing a final pH of 7.8 during thereaction. TS reactions utilizing radiolabeled dUMP were quenched with67 �l of 0.4 N HCl. The quenched reaction solutions were directlycollected into argon-purged Waters Wisp autosampler vials, immedi-ately vortexed, and analyzed by HPLC in combination with radioactiv-ity-flow detection. The substrates and products were then quantified asdescribed below. All samples that were not immediately analyzed werestored at �80 °C until just prior to analysis to minimize degradation. Toconfirm complete quenching of the enzymatic reactions, controls inwhich substrate was added to a premixed solution of enzyme andquench solution was included with each experiment.

HPLC Analysis—The substrates and products were quantified byradio-HPLC using a BDS-Hypersil C18 reverse phase column (250 �4.6 mm, Keystone Scientific, Bellefonte, PA) with a flow rate of 1ml/min. For separation of folates, an isocratic separation using a sol-vent system of 10% methanol in 180 mM triethylammonium bicarbonate

(pH 7.8) was used. The elution times were as follows: H4F, 7.5 min; TSintermediate, 12.5 min; H2F, 14 min, CH2H4F, 16 min. For separationof dUMP and dTMP, an isocratic separation using a solvent system of200 mM triethylammonium bicarbonate was used. The elution timeswere as follows: dUMP, 11 min; dTMP, 18 min. The HPLC effluent fromthe column was monitored continuously using a Flo-One radioactivity-flow detector (Packard Instruments, Downers Grove, IL). The analysissystem was automated using a Waters 712B WISP (Milford, MA)autosampler.

Data Analysis—Rapid chemical quench single turnover and burstdata were fit to single-exponential and burst equations, respectively,using the curve fitting program Kaleidagraph. Stopped-flow measure-ments provided estimates for the association and dissociation rateconstants (kon and koff) and for reaction rate constants. Comparison ofrapid chemical quench and stopped-flow reaction time courses allowedfor the assignment of observed stopped-flow rates to chemical steps orconformational changes.

Spectrophotometric TS Assay—The Km of CH2H4F was determinedusing a steady-state spectrophotometric kinetic assay. T. gondii TS-DHFR enzyme (25 nM) was preincubated with dUMP (100 �M) prior tomixing with CH2H4F (5 to 350 �M), and absorbance was monitored at340 nm using a Hewlett-Packard 8452A spectrophotometer. Initialrates were determined in triplicate using the software provided withthe instrument, and these rates were converted to units of specificactivity using the reported extinction coefficient for the reaction (��rxn

� 6.4 mM�1cm�1). Data in this paper are presented as the average oftriplicate determination with error bars representing the standarddeviation.

Kinetic Simulation—The KINSIM kinetic simulation program wasused to model kinetic data presented in this report (31). The data werefit by a trial and error process, maintaining the constraints of constantsmeasured in this study. The focus of this simulation was to validate theminimal kinetic mechanism elucidated in this study. The model andestimated rate constants are described in Chart 1. Half-sites TS reac-tivity was modeled by defining TS (E) and DHFR (Z) as unique speciesand defining the modeling parameters such that Z � 2E � concentra-tion of TS-DHFR used.

T. gondii Homology Model—A homology model of T. gondii TS-DHFRwas built using the Swiss PDB program in conjunction with the SwissModel homology modeling link available at the Swiss PDB website. TheC-terminal 315 amino acids (residues 295–610) and residues 115–166were modeled using the PDB file for the L. major TS-DHFR structure.The N-terminal 52 amino acids were modeled using the PDB file forPneumocystis carinii DHFR (PDB entry 1CD2), which was the highesthomology DHFR relative to the N-terminal portion of the T. gondiiDHFR domain for which a structure is available.

RESULTS

Overview of the TS-DHFR Reaction—Bifunctional TS-DHFRenzymes catalyze three basic reactions (Scheme 1), the TSreaction, the DHFR reaction, and a bifunctional TS-DHFRreaction. Moreover, each of these reactions can be further sub-divided into two classes of events: binding and dissociation ofligands and those events involved in catalysis. We will firstaddress those events involving binding and dissociation of sub-strates and products, and we will consider an example experi-ment used to measure rates for each. Those events involved in

CHART 1

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chemical catalysis will then be elucidated, including the occur-rence of conformational changes, formation of intermediates,and the identification of rate-limiting steps. We begin with thecharacterization of the DHFR reaction, because it is relativelystraightforward and has features used to help interpret the TSreaction. We will then consider the TS and TS-DHFR reactions,providing a complete TS-DHFR reaction mechanism and newinsight into the mechanism of TS catalysis.

Kinetics of Ligand Binding—The second-order rate con-stants for the binding of ligands to TS-DHFR were determinedby measuring the ligand concentration dependence of the ob-served binding rate. The apparent first-order rates for thebinding of dUMP, dTMP, and H2F to the TS domain and forH2F, H4F, NADPH, and NADP� to the DHFR domain weremeasured using stopped-flow fluorescence. A representativestopped-flow fluorescence trace for the binding of NADPH tothe bifunctional enzyme is shown in Fig. 1a. The trace isbiphasic and fits a double-exponential equation with a fastNADPH concentration-dependent phase of 83.0 3.9 s�1 and aslow NADPH concentration-independent phase of 5.6 0.5s�1. The fast phase represents the apparent first-order bindingrate and conforms to the equation, kobs � kon[L] � koff, in which

kobs, kon, and koff are the apparent first-order observed bindingrate, association rate constant, and dissociation constant, re-spectively. The plot of kobs versus [L] for NADPH binding isshown in Fig. 1b. Accordingly, the binding and dissociation rateconstants are 9.0 0.4 �M�1s�1 and 39.8 3.0 s�1 for theformation and dissociation of the E�NADPH complex, respec-tively, at 25 °C and pH 7.8. The slow NADPH concentration-independent phase is likely to represent a conformationalchange following NADPH binding. A summary of the associa-tion rate constants measured for the various ligands with thebifunctional TS-DHFR enzyme is shown in Table I.

Kinetics of Ligand Dissociation—The rate constants for thedissociation of ligands from TS-DHFR were measured by li-gand competition experiments. A representative stopped-flowtrace for the measurement of the dissociation of H2F from theE�H2F complex using methotrexate as the trapping ligand isshown in Fig. 2. The resulting trace was fit to a single-exponential equation with a rate of 9.0 0.1 s�1, correspondingto the dissociation rate constant, koff, for H2F release from theDHFR domain of TS-DHFR. A summary of the dissociation con-stants obtained from these experiments is shown in Table II.

The DHFR Reaction—The first experiment examining theDHFR activity of the T. gondii TS-DHFR enzyme is a pre-steady-state burst experiment. As shown in Fig. 3a, a burst inDHFR catalysis is observed at a rate of 180 20 s�1. This isfollowed by slow steady-state product accumulation at a rate of

TABLE IKinetics of association of ligands for T. gondii TS-DHFR

at pH 7.8 and 25 °C

Ligand Enzyme species Kon

�M�1s�1 a

NADPH E�H2F 2.9H4F E 27

s�1 b

NADP� E 2.6NADP� E�H2F 2.2NADPH E 9.0H2F E 5.7CH2H4F E 2.0c

dUMP E 13.4dTMP E 10.9H2F E�dTMP�H2FDHFR

d 2.0a The error associated with the determination of each rate constant

is 10% in all cases.b The observed rate of H4F binding was independent of concentration

and likely reflects a conformational change following rapid binding.c Because it was not possible to directly measure the rate of CH2H4F

binding, this rate was estimated based on the CH2H4F concentrationdependence of the TS reaction and by modeling.

d H2F binding to the TS domain was isolated from binding to theDHFR domain by taking advantage of TS ordered binding in whichdTMP binds first. H2F was preincubated with TS-DHFR prior to mixingwith an equivalent of H2F and a large excess of dTMP.

TABLE IIDissociation kinetics of ligands for T. gondii TS-DHFR

at pH 7.8 and 25 °C

Ligand Enzyme species Trapping ligand koff

s�1 a

H2F E�H2F MTX 9.0NADP� E�NADP� NADPH 95NADPH E�NADPH NADP� 7.0NADPH E�NADPH 40b

NADP� E�H4F�NADP� NADPH 7.0H4F E�H4F MTX 39H4F E�H4F�NADP� MTX 17NADPH E�H4F�NADPH NADP� 87CH2H4F E�CH2H4F 40c

dUMP E dTMP 60a The error associated with the measurement of each rate constant

is 10% in all cases.b koff as determined from the binding curve (Fig. 1b). The rate of 7 s�1

for NADPH dissociation from E�NADPH likely reflects the rate of re-lease from a different TS-DHFR conformer than that involved in thebinding experiment.

c The off-rate of CH2H4F was estimated from the product of Kd andKon and by modeling.

FIG. 1. Stopped-flow binding experiment measuring the asso-ciation rate of NADPH to T. gondii TS-DHFR. a, stopped-flowfluorescence trace observed upon mixing TS-DHFR (1 �M) with NADPH(6 �M). b, plot of concentration dependent rate (kobs) versus NADPHconcentration.

FIG. 2. A representative stopped-flow trace for the measure-ment of the dissociation of H2F from the E�H2F complex. Thebifunctional TS-DHFR enzyme (5 �M) was preincubated with H2F (10�M) prior to mixing with excess methotrexate (50 �M).

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5.7 0.6 s�1, corresponding to a rate-limiting step precedingsubsequent turnover. The observed burst amplitude was 31.1 1.4 �M, within the error of the enzyme concentration used,suggesting that essentially 100% of the DHFR sites are active.The second experiment is a single turnover stopped-flow fluo-rescence experiment, the time course for which is shown in Fig.3b. Because enzyme-bound NADPH but not NADP� exhibitscoenzyme fluorescence resonance energy transfer, the timecourse for the fluorescence at 450 nm represents the conversionof NADPH to NADP� at the active site and, hence, the rate ofcatalysis. The data was fit to a single-exponential equationwith a rate (kchem) of 180 2.7 s�1 consistent with the rate ofchemistry observed in the burst reaction. There was no in-crease in the rate of catalysis under conditions in which theenzyme concentration was doubled, indicating that substratebinding was not limiting.

T. gondii DHFR Activation Experiment—Experiments de-signed to examine whether there was domain-domain commu-nication involving DHFR activation in T. gondii TS-DHFR, aspreviously observed with the L. major enzyme, were performed.Both DHFR single turnover and burst experiments were sim-ilar to those described above, except that the enzyme waspreincubated with FdUMP and CH2H4F to form the covalentFdUMP�CH2H4F�TS-DHFR covalent complex. In contrast tothe DHFR activation observed with L. major TS-DHFR (14 to

120 s�1), no significant change in the T. gondii DHFR rate wasobserved (data not shown).

The TS and TS-DHFR Reactions: A Burst in TS Activity?—The first experiment examining the TS activity of the T. gondiiTS-DHFR enzyme was a pre-steady-state burst experiment inwhich enzyme was preincubated with excess [14C]dUMP priorto mixing with a large excess of CH2H4F. The time course forthe reaction is shown in Fig. 4a. The reaction occurs at a linearsteady-state rate with no burst in [14C]dUMP consumption or[14C]dTMP formation. The absence of a burst suggests thatchemistry or a preceding step is limiting in the TS reaction forthis enzyme. A second TS burst experiment was conducted inwhich CH2H4F was the radiolabeled substrate. The time coursefor the consumption of [3H]CH2H4F is shown in Fig. 4b. Incontrast to the linear, steady-state consumption of dUMP, thisreaction occurs with a burst in [3H]CH2H4F consumption. Athird TS burst experiment was conducted using stopped-flowabsorbance. As shown in Fig. 4c, a burst in absorbance at 340nm consistent with the rapid chemical quench burst experi-ment is observed.

The Observation of a TS Intermediate—As shown in Fig. 5(bottom), the burst in TS substrate consumption is coupled tothe formation of a species whose retention time under theHPLC conditions used does not allow for complete separationfrom the product of the reaction, [3H]H2F. Coupling the TS and

FIG. 4. TS burst experiments. a, TS burst experiment in which T. gondii TS-DHFR (25 �M) was preincubated with excess [14C]dUMP (90 �M)prior to mixing with a large excess of CH2H4F (500 �M). b, TS burst experiment in which T. gondii TS-DHFR (40 �M) was preincubated with a largeexcess of dUMP (500 �M) prior to mixing with excess [3H]CH2H4F (120 �M). c, stopped-flow absorbance TS burst experiment in which thebifunctional TS-DHFR enzyme (25 �M) was preincubated with a large excess of dUMP (1 mM) prior to mixing with CH2H4F (500 �M).

FIG. 3. Determination of DHFR cat-alytic rates. a, DHFR burst experimentin which T. gondii TS-DHFR (30 �M) waspreincubated with a large excess ofNADPH (500 �M) before mixing with ex-cess [3H]H2F (100 �M). Time points in theburst phase are the average of triplicatedeterminations with error bars represent-ing the standard deviation. Time points inthe steady-state phase are either singletor duplicate measurements. The inset in ashows an enlarged view of the burstphase. b, DHFR single turnover stopped-flow FRET experiment in which T. gondiiTS-DHFR (50 �M) was preincubated witha large excess of NADPH (250 �M) prior tomixing with limiting H2F (25 �M), and thedecrease in fluorescence at 450 nm wasmonitored over time.

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DHFR reactions allows one to monitor the bifunctional TS-DHFR reaction and thereby monitor the direct conversion of[3H]CH2H4F to [3H]H4F. As shown in Fig. 5 (top), when aTS-DHFR burst reaction is conducted, the burst in[3H]CH2H4F consumption is still coupled to the formation ofthe species seen in the TS reaction. This is followed by slowaccumulation of [3H]H4F without significant accumulation of[3H]H2F.

Further evidence of a TS intermediate was obtained by CCD-array stopped-flow measurements where a shift in the isos-bestic point was observed. The spectra of the E. coli TS reactionunder varying enzyme and substrate concentrations are shownin Fig. 6. The first experiment was conducted under burstconditions: E. coli TS (25 �M) was preincubated with dUMP(500 �M) before mixing with CH2H4F (200 �M). The resultingspectra contained an isosbestic point at 322.8 nm (Fig. 6a). Ashift in the isosbestic point to 337.0 nm was observed when thereaction was repeated using 40 �M CH2H4F (all other condi-tions were identical) (Fig. 6b). Under single turnover conditions(25 �M TS and 10 �M CH2H4F) the isosbestic point was ob-served at 338.1 nm (Fig. 6c). To determine the isosbestic pointfor the quantitative conversion of CH2H4F to H2F, the reactionwas conducted under steady-state conditions in which E. coliTS (2.5 �M) was preincubated with dUMP (500 �M) prior tomixing with CH2H4F (200 �M). The resulting isosbestic pointwas observed at 322.7 nm (Fig. 6d). Similar CCD-arraystopped-flow TS experiments were conducted using bifunc-tional T. gondii and L. major TS-DHFR. Similar shifts in theisosbestic point of the TS reaction spectra for the bifunctionalenzymes were observed (data not shown).

Isolation of TS Catalytic Rates—To isolate the rates of TScatalytic events, a TS-DHFR burst experiment was conducted,the time course for which, is shown in Fig. 7a. The reactionoccurs with a burst in [3H]CH2H4F consumption at a rate of130 10 s�1 coupled to the rapid accumulation of an interme-diate species up to a steady-state concentration correspond-ing to the burst amplitude for the reaction. The burst in

[3H]CH2H4F consumption is followed by a rate-limiting steady-state phase of 6.2 0.6 s�1 in which [3H]H4F is formed withoutsignificant [3H]H2F accumulation.

Evidence for Half-sites TS Reactivity—Another vital piece ofinformation provided by the burst experiment shown in Fig. 7ais the burst amplitude, representing the concentration of TSactive-sites for this reaction. The burst amplitude is 58.9 11.4 �M, or roughly 50% the concentration of TS-DHFR used,and of DHFR active sites, consistent with half-sites TSreactivity.

To examine the CH2H4F concentration dependence of theburst rate and amplitude in the TS burst reaction, a secondseries of TS bursts, similar to that shown in Fig. 4c, wasperformed by stopped-flow absorbance at 340 nm. In this se-ries, the bifunctional T. gondii TS-DHFR enzyme (25 �M) waspreincubated with a large excess of dUMP (1 mM) prior tomixing with excess CH2H4F (40–1500 �M). Plotting the ob-served burst rate versus CH2H4F concentration suggested thatthe relationship was a linear one, consistent with rate-limitingCH2H4F association (kon � 1 �M�1s�1), up to the maximumburst rate of 105 4 s�1 (data not shown) rather than ahyperbolic one that would be consistent with rapid equilibriumand a weak Kd. By contrast, the observed burst amplitudeversus CH2H4F concentration displayed a hyperbolic relation-ship suggesting that the apparent Kd for CH2H4F binding was18 5 �M (Fig. 8a). This is consistent with the steady-state Km

of 17.0 2.0 �M determined for the reaction (Fig. 8b).A TS-DHFR single turnover series was also performed to

determine the rate of the rate-limiting chemical step in the TSreaction (kchem) and to examine the TS-DHFR concentrationdependence on the observed single turnover rate. In this series,the bifunctional TS-DHFR enzyme (12.5–100 �M) was preincu-bated with a large excess of dUMP and NADPH prior to mixingwith limiting [3H]CH2H4F. Fig. 8 (c and d) shows that thereaction time course for this series displays a hyperbolic de-pendence on the concentration of enzyme used. The enzymeconcentration at which the observed rate is half-maximal (Kd

apparent) for this series is 45 8 �M, and the maximum rate(kchem) is 5.5 0.4 s�1.

A Kinetic Model for the T. gondii TS-DHFR Reaction—Themechanistic information obtained in this study was used toformulate a minimal kinetic mechanism for the bifunctionalTS-DHFR from T. gondii (Scheme 3). The TS reaction is depictedin Scheme 3a, and the DHFR reaction is in Scheme 3b. Thismechanism along with the rates obtained in this study was thenused to simulate reaction time courses using the program KIN-SIM. The KINSIM model and rate constants used are describedin Chart 1.The resulting simulations were consistent with exper-imental data for the TS-DHFR reactions (Fig. 7b).

Bursts in L. major TS and TS-DHFR—The TS and TS-DHFR bursts observed in T. gondii TS-DHFR represent amarked mechanistic difference from our previous study ofL. major TS-DHFR. The L. major TS and TS-DHFR reactionswere therefore re-analyzed under identical conditions to theT. gondii reactions described above. The resulting time coursessuggested that CH2H4F binding was rate-limiting under theexperimental conditions employed in our previous study inwhich no burst in TS activity was observed. A representativeTS-DHFR burst time course for L. major TS-DFHR is shown inFig. 9a. This experiment suggests that, like that for T. gondiiTS-DHFR, rapid conversion of CH2H4F to intermediate occursand that, like for T. gondii, this is followed by an overallrate-limiting chemical step associated with dTMP formation.As with T. gondii TS-DHFR, the burst amplitude of the TS-DHFR burst reactions and the steady state concentration ofintermediate that accumulates during the time course are ap-

FIG. 5. HPLC analysis of TS burst reaction: evidence for a TSintermediate. HPLC analysis of the TS (bottom) and TS-DHFR (top)burst reactions. Both reactions were analyzed at t � 100 ms. The peakat 4 min is a contaminant (cont.) formed during chemical synthesis of[3H]CH2H4F.

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proximately equal to one-half the TS-DHFR concentrationused. TS burst experiments were conducted with L. majorTS-DHFR at corresponding concentrations and were consistentwith the TS-DHFR reaction (data not shown).

Bursts in Monofunctional Human and E. coli TS—The ob-servation of a catalytic burst in the TS reaction of the L. majorand T. gondii TS-DHFR bifunctional enzymes also suggested amarked mechanistic difference from previous reports on mono-functional TS enzymes (9). To verify this difference, monofunc-

tional human and E. coli TS enzymes were analyzed underidentical burst conditions to those used for the bifunctionalenzymes. Surprisingly, the time courses for the monofunctionalTS enzymes also displayed a burst in CH2H4F consumptionconsistent with the rapid formation of a TS intermediate pre-ceding a rate-limiting step in TS chemistry. Fig. 9 (b and c)shows the time courses for the monofunctional E. coli andhuman TS enzymes, respectively. As with the bifunctional TS-DHFR enzymes, the monofunctional TS burst amplitudes were

FIG. 7. The TS-DHFR burst reac-tion. a, T. gondii TS-DHFR (120 �M) waspreincubated with a large excess of dUMP(500 �M) and NADPH (500 �M) prior tomixing with excess [3H]CH2H4F (240 �M).The reaction proceeds with a burst in[3H]CH2H4F (�) consumption at a rate of130 10 s�1 and with a burst amplitudeof 58.9 11.4 �M; rapid accumulation ofintermediate (�) at 125 12 s�1 to asteady-state concentration of 62.1 1.4�M; and a slow, steady-state accumula-tion of H4F (●) at a rate of 6.2 0.6 s�1.b, KINSIM model of TS-DHFR burst re-action superimposed on experimentaldata. Experimental: [CH2H4F] (�); [Inter-mediate] (�); [H4F] (●).

FIG. 6. CCD-array stopped-flow spectra. a, E. coli TS (25 �M) was preincubated with excess dUMP (0.5 mM) and mixed with CH2H4F (200�M). The inset is a three-dimensional representation of the data to illustrate the isosbestic point. b, E. coli TS (25 �M) was preincubated with excessdUMP (0.5 mM) and mixed with CH2H4F (40 �M). c, E. coli TS (25 �M) was preincubated with excess dUMP (0.5 mM) and mixed with CH2H4F (10�M). The inset is a magnification of the area surrounding the isosbestic point. d, E. coli TS (2.5 �M) was preincubated with excess dUMP (0.5 mM)and mixed with CH2H4F (200 �M). All reactions were scanned for 2.1 s with a 3-ms integration time. The spectra are shown as viewed down thetime axis. The solid black lines indicate the isosbestic points.

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consistent with half-sites TS reactivity.Homology Model of T. gondii TS-DHFR—To obtain a possi-

ble structural explanation for the difference in DHFR activitiesobserved, specifically the lack of TS-ligand-induced DHFR ac-tivation in T. gondii TS-DHFR that is observed in L. major, ahomology model of T. gondii TS-DHFR was built as describedunder “Materials and Methods.” As shown in Fig. 10, the ho-mology model suggests that the T. gondii TS-DHFR enzyme

lacks an N-terminal tail, linking the DHFR and TS domains,that is present in the crystal structure of L. major TS-DHFR.

DISCUSSION

In this work, we have characterized the complete kineticscheme for bifunctional TS-DHFR from T. gondii. In addition toproviding the detailed enzymatic mechanism for this importantchemotherapeutic target, this study compares the mechanism

FIG. 8. Evidence for half-sites TS re-activity. a, hyperbolic dependence of theburst amplitude on CH2H4F concentra-tion for a series of stopped-flow burst ex-periments. b, steady-state Km determina-tion for CH2H4F (Km � 17.0 2.0 �M). c,TS-DHFR single turnover series in whichT. gondii TS-DHFR (12.5 �M (f), 25 �M

(●), 50 �M (Œ), or 100 �M (�)) was prein-cubated with a large excess of dUMP (500�M) and NADPH (500 �M) prior to mixingwith limiting [3H]CH2H4F (5.6 �M). d, hy-perbolic dependence of the observed sin-gle turnover rate on enzyme concentra-tion (Kd apparent � 45 8 �M). For a andb, error bars represent the standard devi-ation of triplicate determination. For d,error bars represent the error associatedwith each single-exponential fit.

SCHEME 3

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with that from bifunctional TS-DHFR from L. major for whicha structure is known, highlighting both similarities and differ-ences in mechanism. Finally, the use of rapid transient kineticsmethods in this study provides the first direct kinetic evidencefor half-sites TS reactivity and for the accumulation of anintermediate during TS catalysis.

An Overview of the T. gondii TS-DHFR Reaction—For theT. gondii TS reaction (Scheme 3a), the overall rate-limitingstep occurs during chemistry at a rate (kchem) of 5.5 s�1. This isconsistent with previous studies with other TS enzymes, whichhave suggested that a hydride transfer step occurring in thefinal step of TS catalysis is rate-limiting (9). For the T. gondiiDHFR reaction (Scheme 3b), chemistry occurs at a relativelyfast rate (kchem) of 180 s�1, compared with an overall rate-

limiting conformational change (kss) at a rate of 5.6 s�1 thatoccurs immediately after NADPH binding.

Comparison of L. major and T. gondii DHFR Mecha-nisms—As might be predicted on the basis of relative sequencehomology, the differences between L. major and T. gondii TS-DHFR reside primarily in the DHFR mechanisms. The firstmajor difference is the lack of DHFR activation in the T. gondiienzyme. The homology model of T. gondii, when compared withthe crystal structure of L. major, suggests a possible explanationfor this difference. We postulate that the 23-amino acid tail ofL. major that is absent in the T. gondii homology model mayserve to inhibit DHFR activity in a TS conformation-specificmanner. This domain-domain communication mechanism mayserve as a TS activity sensor activating DHFR activity duringTS catalysis, further coupling the sequential TS and DHFRactivities.

A second notable difference between the L. major and T.gondii DHFR mechanisms is found in the location of the rate-limiting step for each. In both cases, the rate-limiting stepinvolves a conformational change; however, this step occurs indifferent places along the respective DHFR pathways. Asshown in Scheme 4, the rate-limiting conformational changefor the T. gondii enzyme occurs after NADPH binding, whereasit takes place after NADP� release from E�H4F�NADP� in theL. major enzyme.

A final subtle, but noteworthy, difference between the DHFRmechanisms for these species is that the product release path-way for L. major DHFR is kinetically restricted to one path,whereas product release in T. gondii can occur via multiplekinetically competent paths (Scheme 4). This is not to say thatmultiple product release pathways are not utilized by theL. major enzyme, but that only one of the pathways is kineticallycompetent in that it contains no one rate slower than the overallrate-limiting step for the enzyme. In contrast, all the possibleproduct release pathways in the T. gondii enzyme are kineti-cally competent and therefore likely to be equally utilized.

Evidence for Half-site TS Reactivity—It has been suggestedthat TS is a half-sites-reactive enzyme in which only one TSmonomer of dimeric TS is catalytically active at a time. Thisproposal is based on structural evidence that suggests that TSbinds ligands asymmetrically to each monomer’s active site andmutagenesis studies in which an active site and non-active siteTS dead mutant are combined to form a heterodimer with fullyrestored TS activity (17–19). To our knowledge, however, thisstudy provides the first direct kinetic evidence for half-sitesreactivity in TS enzymes.

FIG. 9. L. major, E. coli, and human TS burst reactions. a, L. major TS-DHFR burst (52.6 3.8 �M amplitude) in which TS-DHFR (120 �M)was preincubated with a large excess of NADPH (500 �M) and dUMP (500 �M) prior to mixing with [3H]CH2H4F (240 �M). b, E. coli TS burst (10.6 0.8 �M amplitude) in which E. coli TS (30 �M) was preincubated with large excess of dUMP (500 �M) prior to mixing with [3H]CH2H4F (60 �M).c, human TS burst (13.7 0.6 �M amplitude) in which TS-DHFR (30 �M) was preincubated with large excess of dUMP (500 �M) prior to mixingwith [3H]CH2H4F (60 �M).

FIG. 10. T. gondii homology model. A homology model of T. gondiiTS-DHFR (bottom; blue) was built as described under “Materials andMethods.” Comparison of this model with the crystal structure of L.major TS-DHFR (top; red) demonstrates that a 23-amino acid N-termi-nal tail linking the DHFR and TS domains in the L. major structure(tail shown in yellow) is absent in the T. gondii structure (N-terminalthree amino acids shown in yellow).

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The pre-steady-state burst amplitude observed for the TSreaction in T. gondii TS-DHFR, which reflects the concentra-tion of TS active sites, is approximately one-half the concen-tration of TS used, whereas the corresponding DHFR burstexperiment suggests that the enzyme contains essentially100% DHFR active sites. In addition, analysis of the concen-tration dependence of TS activity under single turnover andburst conditions suggests that this half-sites reactivity is aresult of asymmetric substrate binding. Specifically, the obser-vation that the apparent Kd for the single turnover series inwhich the enzyme is in excess and determines binding is ap-proximately twice the Kd for the burst series in which CH2H4Fdetermines binding suggests that only one-half of the TS pres-ent in solution can productively bind CH2H4F. Taken together,these studies provide the first direct kinetic evidence in supportof half-sites TS reactivity, in which only one TS monomer bindssubstrate productively.

It is worth noting that a burst amplitude corresponding to50% enzyme concentration might also be explained by twoother scenarios: (i) 50% misfolded enzyme or (ii) a 50–50 equi-librium between two forms of the enzyme prior to chemistry.The former is unlikely, because a 50% burst amplitude wasobserved with multiple enzyme preps and with TS from variousspecies, and the latter scenario is unlikely on the basis of theresults of the current as well as previous studies, which areconsistent with half-site reactivity resulting from asymmetricbinding of TS ligands.

Evidence for a TS Intermediate—It has also been proposedthat TS catalysis involves the formation of an iminium ion formof CH2H4F and that this is the reactive form of the cofactor.Moreover, there is structural evidence involving a mutant of TSlacking a C-terminal valine crystallized with CH2H4F and

FdUMP in which (6R)-L-5-hydroxymethyltetrahydrofolate(HO-CH2H4F) was found to be bound at the active site suggest-ing the formation of the putative iminium ion during TS catal-ysis (10). However, this study provides the first direct kineticevidence for the accumulation of a TS intermediate during TScatalysis.

The presence of a burst in substrate (CH2H4F) consumptionwithout a corresponding burst in product formation and theobservation of a rapidly formed transient species not attribut-able to substrate or product in HPLC analyses provide the firstdirect kinetic evidence for the accumulation a TS intermediate.

The accumulation of a TS intermediate is further supportedby the shift in the isosbestic point for the TS reaction in thestopped-flow CCD experiments. Specifically, under steady-state conditions or burst conditions where CH2H4F is in largeexcess over enzyme, a single isosbestic point is observed at�322 nm for the TS reaction. Under conditions where enzymeconcentration becomes significant relative to CH2H4F, such asunder single turnover conditions, there is a shift in the isos-bestic point to �337 nm. Stadman et al. (32) have shown thatthe wavelength of an isosbestic point may change under vary-ing experimental conditions provided (i) either the molar ab-sorptivity of the substrate changes under the varying experi-mental conditions or (ii) the fraction of the substrate that isconverted to multiple products changes. The observed shift inthe isosbestic point during the TS reaction is consistent withthe latter, in which there is formation of an enzyme-boundintermediate species that becomes increasingly significant asthe reaction conditions approach single turnover conditions.

Considering the proposed TS mechanism (Scheme 2), theresults of previous TS mechanistic studies, and the results ofthis study, we suggest that this TS intermediate is the putative

SCHEME 4

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iminium ion form of the cofactor. Specifically, there is a burst inCH2H4F consumption without a corresponding burst in dUMPconsumption, and previous kinetic isotope effect studies with E.coli TS suggest that either the CH2H4F cofactor or its iminiumion form, but not any other intermediate species, accumulatesat the active site during TS catalysis (9). On the basis of thesekinetic isotope effect studies and the proposed mechanism, it islikely that the rate of iminium ion formation is limited by theconformational change represented by step 1 in Scheme 2.

Half-sites TS Reactivity and Intermediate Formation inL. major TS-DHFR and Monofunctional TS Enzymes—Thedirect observation of half-sites TS reactivity and the formationof a transient TS intermediate were novel observations. Ac-cordingly, it was of interest to see whether similar kineticswould be observed in other TS enzymes. Analysis of the bifunc-tional TS-DHFR from L. major and of monofunctional E. coliand human TS suggest half-site reactivity and intermediateformation also occurs with TS from these species.

Although this study has provided new insight into theT. gondii TS-DHFR mechanism and into the TS mechanism ingeneral, several questions remain unresolved. First, it remainsto be established whether T. gondii TS half-sites reactivity maybe coordinated, with the activity alternating between mono-mers of a TS dimer, or whether this activity is uncoupled andrandom. An additional possibility as suggested by studies withthe R126E/C146W heterodimeric mutant E. coli TS enzyme isthat only one subunit is required for activity with the othersimply serving as a scaffold for the active subunit (18). Second,although DHFR activation is not observed with T. gondii TS-DHFR, a detailed study of potential domain-domain communi-cation in this enzyme remains to be conducted. It is possiblethat more subtle communication, such as changes in substrateaffinity (cooperativity), between the TS and DHFR domainsmight occur in this enzyme. Finally, it would be of interest tocompare the detailed T. gondii TS-DHFR mechanism to that ofhuman TS and DHFR. Subtle differences, such as the identityand location of rate-limiting steps or conformations unique tothe bifunctional enzyme, might provide crucial insights for thedevelopment of novel therapeutics specific for T. gondii.

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Eric F. Johnson, Wolfgang Hinz, Chloé E. Atreya, Frank Maley and Karen S. AndersonAND TS HALF-SITES REACTIVITY

(TS-DHFR)-Dihydrofolate Reductase: EVIDENCE FOR A TS INTERMEDIATE Thymidylate SynthaseToxoplasma gondiiMechanistic Characterization of

doi: 10.1074/jbc.M206523200 originally published online August 20, 20022002, 277:43126-43136.J. Biol. Chem. 

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