mode of binding of folate analogs to thymidylate synthase

T H E JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 3, Issue of January 21, pp. 1873-1882, 1994 Printed in U.S.A.

Mode of Binding of Folate Analogs to Thymidylate Synthase EVIDENCE FOR TWO ASYMMETRIC BUT INTERACTIVE SUBSTRATE BINDING SITES*

(Received for publication, April 21, 1993, and in revised form, September 21, 1993)

Indeji t K. DevS, Walter S. Dallas, Robert Ferone, Mary Hanlon, David D. McKee, and Barbara B. Yates From the Division of Molecular Genetics and Microbiology, Burroughs Wellcome Company, Research Diangle Park, North Carolina 27709

Human thymidylate synthase is a polymeric protein composed of two subunits with identical primary structures. In this study we determined the binding affinities of S,10-methylene tetrahydropteroyltetraglutamate (folate substrate) and a group of close structural folate analog inhibitors. Thymidylate synthase bound both mono and polyglutamylated folate substrates and analogs more tightly in the presence of deoxyuridylate. These results and product inhibition studies confirmed that the orders of substrate addition and product release from thymidylate synthase were similar for mono and polyglutamylated substrates. Equilibrium dialysis studies showed that the folate substrate in a ternary complex with deoxyuridylate bound to one of the subunits (site A) with a & of 720 MI. The binding of the substrate to the second subunit (site B) was much weaker, and the& could not be determined by this method. How- ever, dissociation constants for each subunit could be measured for the folate analog inhibitors, and, depending on the inhibitor, the relative & value for each subunit varied substantially. For example, formyl-6,8-dideazafolate and tetraglutamylated lO-propargyl-S,8- dideazafolate bound to both sites with similar & values, whereas D1694Gl~ bound to subunit A with a higher affinity (& = 1.0 MI) than to subunit B (Kd = 30 MI). In contrast, 1843U89 (mono or diglutamylated form) had a much higher affinity for subunit B (& ”0.1 MI) compared with subunit A (& -400 m). Enzyme inhibition kinetic analyses showed that the Kt values of 1843U89 were quite low (0.1 MI) and that the inhibition was noncompetitive. In contrast, the other folate analogs inhibited the enzyme via mixed inhibition (Le. both the K, for the folate substrate and the V,, were altered). We conclude that the two subunits of thymidylate synthase bind folate substrates and analogs differently and that the asymmetric binding of the ligands is the major factor that determines the inhibition kinetics of each folate analog inhibitor.

Thymidylate synthase (TS1; EC 2.1.1.45) catalyzes the re-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. Tel.: 919-315-4314;

The abbreviations used are: TS, thymidylate synthase; dUMP, 2‘-

uridylate; CB3717 or PDDF, lO-propargyl-5,8-dideazafolate; FDDF, 10- deoxyuridylate; dTMP, thymidylate; FdUMP, 5-fluoro-2‘-deoxy-

formyl-5,8-dideazafolate; IC1 D1694, N-(5-[N-(3,4-dihydro-2-methyl-4- oxoquinazolin-6-ylmethyl)-N-methylamino]2-thenoyl)-~-g~utamic acid; BW1843U89, ~S~-2-(5-(((1,2-dihydro-3-methyl-l-oxobenzo[~quinazolin- 9-yl)methyl)amino)-l-oxo-2-isoindolinyl)glutaric acid; HPLC, high per- formance liquid chromatography; GPLC, gel permeation liquid chroma-

Fax: 919-315-0656.

tography.

ductive methylation of deoxyuridylate (dUMP) to thymidylate while converting the cofactor 5,lO-methylene-H4PteGlu to H2PteGlu. Various polyglutamylated forms of the cofactor (H,PteGlun) can act as the one-carbon donor and reductant (for review see Refs. 1 and 2). X-ray crystallography of TS from a variety of sources (human, Lactobacillus casei, T4, Escherichia coli) has shown that in the absence of ligands, the enzyme is a symmetrical homodimer with two active sites (1-5). However, the enzyme in a binary complex with either mono or polyglutamylated lO-propargy1-5,8-dideazafolate (PDDF or CB3717) or in a ternary complex with PDDF and either dUMP or 5-fluoro-2’-deoxyuridylate (FdUMP) showed small but significant differences in ligand binding for each monomer (3-6).

BW1843U89, PDDF, and IC1 Dl694 are potent inhibitors of TS which have been tested or are currently being developed as antitumor agents (7-10). Some experimental results concern- ing the mechanism of inhibition of TS by these folate analogs are contradictory. For example, x-ray crystallographic data indicate that PDDF occupies both active sites of TS with the same orientation as the folate substrate (11). In contrast, kinetic studies suggest that PDDF and other folate analogs bind inde- pendently of either folate or nucleoside substrate (2, 12-16). Different explanations have been proposed to explain this para- dox. Santi and Danenberg (2) suggested that these inhibitors are actually the analogs of the product H2PteGlu which, based on ordered mechanism of substrate addition and product release, are expected to give noncompetitive kinetics. Pogolotti et al . (17) observed that a reversible ternary complex of PDDF, L. casei TS, and dUMP slowly undergoes isomerization to a more stable complex with a concomitant change from competitive to noncompetitive kinetics. More recently, Keyomarsi and Moran (18) have proposed that PDDF metabolites inhibit both active sites of dimeric TS by binding tightly to one site. Based on this model, Ward et al. (19) interpreted the results of their studies on mouse TS inhibition by D1694Glu4 as mixed noncompetitive inhibition.

In this report, we determined the binding affinities of the folate substrate and six folate analogs to each subunit of dimeric TS by equilibrium dialysis, ultrafiltration, gel permeation chromatography, and kinetic enzyme assays. The relative binding affinities of the folate substrate and four of the analogs for each subunit varied substantially, and depending on how an inhibitor bound to each subunit in relation to the substrate, it resulted in either noncompetitive or mixed inhibition kinetics of TS.

EXPERIMENTAL PROCEDURES Materials-Monoglutamylated PDDF and [l4C1PDDF (60 cpdpmol)

were kindly supplied by M. G. Nair (University of Southern Alabama, Mobile). Monoglutamates of 1843U89 and Dl694 were synthesized by W. Pendergast and S. Dickerson. BW1843U89 was tritiated by tritium gas exchange (Moravek Biochemicals) and was purified by diethyl ami- nopropyl column chromatography (catalog 623101, Varian Bond Elut).

1873

1874 Folate Analog Binding to Thymidylate Synthase The column was preequilibrated with 10 m NH,HC03, and 850 nmol of 1843U89 was applied to the column. The column was washed with methanol and 500 m~ NH4HC03, 2.0 ml each. r3H]1843U89 (30 Ci/ mmol) was eluted in 2 ml of 50% acetonitrile in 500 m~ NH,HCO,, dried, and redissolved in 500 pl of 50% ethanol. The solution was clari- fied by centrifugation and stored in the dark at 4 "C.

Polyglutamates of the folate analogs were synthesized using purified folylpolyglutamate synthetase isolated from E. coli (20). Reaction mixtures contained 50 m~ Tris-HCI, pH 8.9,5 m ATP, 10 m MgC12, 50 m~ KCI, 0.1% bovine serum albumin, and 250 units of folylpolyglutamate synthasdml. The substrate concentrations were adjusted to produce the desired products. They were either 16 p~ r3H]1843U89 or 0.50 m~ Dl694 with 20 m~ sodium glutamate; 20 p~ Dl694 or PDDF with 0.2 mM sodium [3Hlglutamate (4 pCi/nmol); 0.5 m~ PteGluz with 2 m~ sodium [14Clglutamate (12.5 pCi/pmol). The solutions were filter ster- ilized (Millipore Millex GV, 0.22 pm) and incubated at 37 "C in the dark. The progress of the reaction was followed by HPLC on a C18 Novapak column (radial compression, Waters, Boston) with an appropriate acetonitrile gradient in 5 m~ tetrabutylammonium phosphate, 10 m~ ammonium phosphate, pH 7.0. When the desired polyglutamylated product predominated (times vaned between 0.5 and 120 h for different analogs), the synthesis was terminated by boiling the reaction mixture for 5 min. Reaction mixtures were cooled and centrifuged, and the pH was adjusted to pH 5.5 with 0.1 N acetic acid.

Samples were applied to an HPLC column (C18 pBondapak column, 19 x 150 mm, Waters) at 2.0 mumin, and the column was washed with 500 ml of 50 mM ammonium acetate, pH 5.5, a t 9.9 mumin. The polyglutamates were eluted with a linear gradient of 6 2 0 % acetonitrile in 50 m ammonium acetate, pH 5.5, delivered over 60 min. PDDFGlu,, 1843U89Glu2, D1694Glu4, and PteGlu, eluted at 13, 16, 10, and 0.25% acetonitrile, respectively. The purified samples were lyophilized and redissolved in HzO. An aliquot of each final product was examined by HPLC. Concentrations were determined by comparison with standard curves and were confirmed by W spectral analyses (Perkin Elmer Lambda 6). Each compound was better than 98% pure as determined by HPLC analyses.

The [14C1PteGlu4 (55 p ~ ) was reduced to (6s)- [14ClH4PteGlu4 with NADPH (165 p) and bovine liver dihydrofolate reductase as described previously (21). The product was purified on a C18 Novapak column with a linear gradient of 3-10% acetonitrile in 0.1% trifluoroacetic acid (2 d m i n , 15 mid. [14C1H4PteGlu4 eluted at 14.6 min and was collected into 2-mercaptoethanol (50 mM final concentration). The sample was dried, redissolved in 50 m~ Tris-HC1, pH 8.0, 0.2 M 2-mercaptoethanol, and stored at -80 "C. An aliquot was examined on HPLC to determine purity and concentration. A 50-fold molar excess of formaldehyde was added to convert [14ClH4PteGl~4 to (6R)- 5,10-[14Clmethylene- H4PteGlu4. The concentration of 5,10-[14Clmethylene-H4PteG1U4 was confirmed by TS assay. 5,10-[14ClMethylene-H4F'teGlu4 was better than 97% pure as determined by HPLC analyses.

Cloning and Expression in E. coli ofthe Human TS cDNA-A h g t l l (22) cDNAlibrary generated from SV40-transformed human WI-38 cells was obtained from Dr. Barbara Wold (23). Oligonucleotides were synthesized (model 8700 Milligefliosearch, Burlington, M A ) as probes to screen for the appropriate phage and were copied from the sequence for a human TS cDNA reported by Takeishi et al. (24). The phage-screening procedure followed the protocol outlined by Maniatis et al. (25). Only one probe-positive plaque was identified from the lo7 plaques that were screened, and this plaque was purified further and phage DNAisolated (25). The phage DNA was cut with EcoRI, the enzyme used for the library construction, and the DNAfragments were separated by agarose gel electrophoresis. The phage was found to have two EcoRI inserts of 1,200 and 750 base pairs. Only the larger fragment was found to hy- bridize with the oligonucleotide probes (data not shown). The larger fragment was then subcloned into pBS, forming pHTS29 (26). The DNA sequence of the large DNA fragment was determined (27) and compared with the sequence reported by Takeishi et al. (24). The TS cDNA sequence was reported to have an EcoRI site 126 base pairs from the initiation codon, and these 126 base pairs were found to be missing from our cDNA isolate. The sequence we determined for our partial cDNA isolate matched the sequence that had been reported.

To overexpress human TS in E. coli, we needed to synthesize the missing portion of the cDNA and then insert the entire coding sequence into an expression vector. Synthesizing and cloning the missing cDNA was problematic for us. The missing bases were synthesized as two fragments. The first fragment to be cloned included an HindIII site 5' to the initiation codon and terminated in a PstI site that was present in the full-length cDNA. This double-stranded synthetic oligonucleotide was cloned into pBS+ to form pTS004. A second synthetic oligonucleo-

tide was made which included the PstI site and extended to the EcoRI site. This oligonucleotide was cloned into pTS004, forming pTSOl2. All isolates from this experiment were found to have a single base mutation that would substitute an amino acid in the gene product. Therefore, a third oligonucleotide was made which extended from an SphI site to the PstI site. The oligonucleotide was cloned into pTSO12, forming pTS050. This plasmid had the missing part of the human TS cDNA.

To construct the expression vector, the vector itself was modified before the cDNAwas inserted. The vector we chose has the T7 promoter on a high copy number plasmid that encodes tetracycline resistance (28). The vector was first modified to have unique HindIII and EcoRI sites for directional cloning of the cDNA for transcription from the T7 promoter. The vector was designated pTX008. The large cDNA fragment was cloned from pTS029 as an EcoRI to HpaI fragment and inserted into pTX008 that had been partially digested with BamHI, treated with T4 DNA polymerase to make the free ends base paired, and cut with EcoRI. The resulting plasmid was designated pTS001. The cloned synthetic oligonucleotide from pTSO5O was cloned into pTSOOl as an Hin- dIII to EcoRI fragment forming pTSO6O. This plasmid was then transformed into TX61, which is a TS mutant of the T7 expression strain BL21[DE3](29,30). The strain is known to allow significant expression of cloned genes without induction. However, pTSO6O did not enable TX61 to grow in the absence of an exogenous source of thymidine. We had noted that the start of the coding region of TS was GC-rich, and the DNA sequencing gels were compressed in this region. We speculated that either our reading of the sequencing gels was in error and our synthesized sequence was not as we had intended, or that the GC-rich region might compromise, in some way, the initiation of translation. Therefore, we used site-directed mutagenesis to modify the bases of several codons at the start of the TS coding region and replaced G or C residues with T residues: AAGCTT ATG CCT GTGtT) GCC(T) GGC(T) (31). The new plasmid, pTS080, was found to complement the thyA mutation of TX61.

Purification of Human TS-Human TS from HeLa cells and E. coli TS was purified according to the methods of Rode et al. (32). Recombi- nant human TS was isolated from E. coli strain TX61(pTS080). This strain was grown in Luna broth with 10 pg/ml tetracycline, 20 pg/ml kanamycin, and 0.2% lactose. Cell-free extracts from 200 g, wet weight, of cells were prepared by methods described earlier (33). Human TS was purified according to published procedures (32) except the enzyme was eluted from an aflinity column of lO-formy1-5,8-dideazafolate-Sepha- rose (2.5 x 10 cm) with 2 liters of buffer containing 1.0 M KC1 and 2.0 M urea. No Triton X-I00 was added to the wash and elution buffers. The active fractions were pooled (800 ml) and concentrated; urea and KC1 were removed quickly by tangential flow diafiltration using 20-kDa Sartorius cellulose acetate filters and dialysis against bufferA(32). The concentrated and dialyzed sample was applied to a DEAE-cellulose column and eluted with a linear gradient of buffers C and D (32). The active fractions were pooled and precipitated with 70% (NH4)2S04, dialyzed, and stored in 0.05 M Tris-HC1, pH 7.5,0.02 M 2-mercaptoethanol, 0.25 M KCI, 0.05 M MgC12, 5% glycerol a t about 20 mg/ml. The yield was about 150 mg of 9599% pure enzyme from 200 g of cells. The enzyme was stable for more than a year when stored at -80 "C.

Binding of Radiolabeled Ligands to TS by Equilibrium Dialysis, Ultrafiltration, and Gel Permeation Chromatography-Equilibrium dialysis was carried out in 0.05 M Tris-HC1, pH 7.5, 0.02 M 2-mercaptoethanol, 0.25 M KCI, 0.05 M MgClz at 4 "C in a multicavity, 0.5-ml microdialysis cell (Bel-Art Products, Pequannock, NJ). The methods for the preparation of dialysis membrane and estimation of concentration of free ligand [I] and enzyme-ligand complex [E11 were as described by Pattishall et al. (34). The enzyme concentration varied between experiments but was in the range of ICd values. Human TS at a final concentration of 100 n~ and higher was stable throughout the equilibration period. However, about 15% of the enzyme activity was lost when 40 nM human TS was used, and data were corrected for this loss in activity. The loss in enzyme activity was much greater at concentrations below 40 nM. Therefore, binding studies with the ternary complex of [3H]1843U89Glul.human TS.dUMP were performed using gel permeation chromatography (35). The reactions (0.2 ml, containing 1.8 nM enzyme) were prepared with freshly diluted enzyme and applied to a Fast-desalting column (fast protein liquid chromatography, 10 x 10 cm, 1 mumin) preequilibrated in the equilibrium dialysis buffer to separate free inhibitor from the enzyme-inhibitor complex (35).

The ternary complex of human TS.dUMP~5,10-[14Clmethylene- H4PteGlu4 was converted to products during equilibrium dialysis at 4 "C. Therefore, reaction mixtures (0.5 ml) containing 5.0 ITLM HCHO, 20 m~ 2-mercapt~ethano1, and 0.5 mM dUMP were prepared in equilibrium dialysis buffer a t 4 "C and rapidly transferred to an ultrafilter (What-

Folate Analog Binding to Thymidylate Synthase 1875 man; 30,000 molecular weight cutoff, catalog 6610-7196). Centrifuga- tion for -60 s produced about 0.05 ml of filtrate containing the free ligand. Less than 5% of the substrate was converted to the products during ultrafiltration. The concentrations of HCHO and 2-mercaptoeth- an01 have no effect on the enzyme activity. The concentration of free ligand and the sum of free ligand plus the enzyme-ligand complex were quantitated from radioactivity measurements of the filtrate and reten- tate, respectively. The concentration of the enzyme-ligand complex was calculated by the difference, and the data were analyzed by a transformation of the Scatchard equation (36) given by Scott (37)

[IlI[E.Il = K,/(n[EI) + [Ill(n[El) (Eq. 1)

where [I], [E], and [E.I] are the concentrations of free inhibitor, total enzyme, and enzyme-inhibitor complex, respectively, and II is the mol of ligand-binding sitelmol of dimeric enzyme. Linear regression of [IJ/[E.Il uersus [I] gives a slope of l/(n[El) and intercept on the abscissa of -Kd. A nonlinear plot of [III[E.I] versus [I1 is indicated if multiple independent classes of binding sites are present, and then Kd values were calculated using the segregation procedure described by Pattishall et al. (34).

A competitive binding assay was used to calculate Kd values for ligands that bind to the same site (34). In this assay the Kd value of a nonradioactive ligand (Kd2) was calculated from the concentration of ligand which reduces the formation of the enzyme-radioactive complex by 50% (I5o) together with the known concentration of the radioactive ligand [SI and the previously determined Kd value (Kd2) of the radioactive ligand according to a modified transformation of the Michaelis- Menten equation.

K d 2 = I d 1 + [Sl/Kd,I (Eq. 2)

Association and Dissociation Rate Constants of Nonproductive Com- plexes ofHuman TS-Garvey and Santi (35) have used gel permeation chromatography (Sephadex G-25) to study the kinetics of the formation of nonproductive complexes of TS. Reactions (0.1 ml) were carried out in 0.05 M Tris-HC1, pH 7.5, 0.02 M 2-mercaptoethanol, 0.15 M KC1, 0.5 mg/ml bovine serum albumin, 1,400 nM 6-[3HldUMP (15 Ci/mmol, Moravek Biochemicals), 30 n~ human TS, and various amounts of the folate analogs at 25 "C. The rate of dissociation of the binary complex of human TS.[3HldUMP or dUMP from a ternary complex was studied by the addition of a 1,000-fold excess of unlabeled dUMP to the preformed complexes. Aliquots (0.05 ml) of the reactions were applied to a gel permeation HPLC column (TSK G3000,7.5 x 600 mm) and eluted at 1.0 mumin to separate the enzyme-bound C3H1dUMP from the free [3HldUMP in about 20 min. The apparent rate constants of association and dissociation were calculated according to the procedures outlined by Pogolotti et al. (17).

Enzyme Assays-Initial velocities for TS were determined in 0.15 M Tris-HC1, pH 8.0, a t 37 "C using either the tritium release assay of Roberts (38) as modified by Dev et al. (39) or by a spectrophotometric assay (40). Reactions were started by adding TS to a final concentration of 1-3 and 40 nM in the radioactive and the spectrophotometric assays, respectively.

Because of possible interference by the slow isomerization of TS (17), kinetic analysis of the inhibition of human TS by folate analogs was investigated by a continuous enzyme assay, and the initial rates within 1.0 min of the start of the enzyme assays were considered. However, in the case of some of the tight binding inhibitors (K, < 2 IIM) the tritium release assay was used to determine K, values. Relative values of K, were obtained by normalizing with the K, = 1.0 for each inhibitor determined at the lowest concentration (0.25 w) of the substrate used. In the inhibited reactions, sufficient drug was used to give 50-95% inhibition over a 1-2-min time period. The inhibited reactions were linear thoughout this time interval. The data were evaluated by the method of Henderson (41) for tight binding noncompetitive inhibitors according to Equation 3 and for tight binding competitive inhibitors by Equation 4,

IJ(1 - v;/u,) = K,(v,/u,) +E, (Eq. 3)

where I, is the total inhibitor concentration, v, is the velocity of the uninhibited reaction, ui is the inhibited velocity, K, is the inhibitor constant, and Et is the total enzyme concentration. Plots ofIJ(1 - u,/v,) versus UJV, result in straight lines with slopes of K, and ordinate intercepts of E,

IJ(1 - U;/U,,) = q(l + ~/K,,,)(LJ,/u,) +E, (Eq. 4)

where S is the concentration of competing substrate, 5,lO-methylene-

TABLE I Dissociation constants and n values of dUMP for dimeric TS with

different additions to equilibrium mix

Additions Kd n

PM

None 7.5 & 1.4 (5)" 0.9 f 0.1 FDDF 3.5 1.3 (2) PDDF

1.6 f 0.3

1843U89 2.3 2 0.6 (2) 2.5 2 0.7 (3) 1.25 f 0.1

1.5 f 0.2

a The number of separate determinations is given in parentheses. Values are expressed as mean S.E.

H4PteGlu,, and K,,, is the Michaelis constant of the substrate. Plots of IA(1 - vi/v,,) uersus VJV, result in straight lines with slopes of K, (1 + FYK,,,) and ordinate intercepts of E,. Note that the slope of these lines will be independent of [SI for strictly noncompetitive inhibition, but will increase with increasing [SI for competitive or mixed inhibition. Data were analyzed by using the QUADFIT program of Henderson (41) or, when the program failed to converge, linear regression. V,, and K,,, parameters from all assays were derived using a computer program that performed a weighted nonlinear square regression analysis of data by using the Michaelis-Menten equation (42).

RESULTS

Purification and Characterization of HeLa and Recombinant Human TS-Human TS from the two sources was purified to near homogeneity. The proteins from both the sources migrated in sodium dodecyl sulfate-polyacrylamide gels with a molecular mass of about 35 kDa. Steady-state kinetic parameters for the recombinant human TS were K , (dUMP) = 2.5 p ~ , K, ((6R)- 5,10-methylene-H4PteGlulf = 12 p ~ . The specific activity of different preparations varied between 0.8 and 1.2 pmol/min/mg of the recombinant human TS at 37 "C. The kinetic parameters of the recombinant human T S were very similar to HeLa TS (data not shown) and the published values of human TS (43- 45). Four different folate analogs inhibited the TS from the two different sources with similar inhibition constants (data not shown). The number of active sites was determined by titration of recombinant human TS with FdUMP by charcoal assay (44) with 5,lO-methylene-H4PteGlu4 as the folate substrate; the number of active sites was 1.7 active siteddimer, suggesting that about 85% of recombinant human TS was active in our preparation.

Purified human TS from HeLa cells and recombinant E. coli was subjected to amino-terminal amino acid sequence, and the first 32 residues were found to be identical, with the first resi- due being methionine. The amino-terminal amino acid sequence also matched with the amino acid sequence deduced from the published nucleotide sequence (data not shown; 24). The comparison showed that the two proteins were identical. Unlike human TS from other sources (43), the amino terminus of human TS from HeLa cells and recombinant E. coli was not blocked. The data suggest that the recombinant human TS has catalytic and physical properties similar to the authentic enzyme isolated from HeLa cells. The recombinant human TS was used for the following binding and kinetic studies.

Binding of Substrates dUMP and (6R)-5,10-methylene- H4PteGlu4 to human TS-Measurements of the binding of L3H1dUMP to TS in a binary complex by equilibrium dialysis yielded a Kd value of 7.5 p~ with n = 0.90 for dimeric human TS (Table I). The addition of FDDF, PDDF, or 1843U89 to form the ternary complex resulted in a 1.4-1.8-fold increase in the stoichiometry of dUMP bound per mol of TS and a 2-3-fold reduc- tion of the Kd for dUMP (Table I).

The binding of 5, 10-methylene-H4PteG1u4 to human TS in a binary complex, or with dUMP, in a ternary complex was measured by equilibrium dialysis using an enzyme concentration of 4 p~ and 5,10-[14Clmethylene-H4PteGlu4 concentrations up to 5 p ~ . The results presented in Fig. 1 show weak binding of the

1876 Folate Analog Binding to Thymidylate Synthase

Free [SI. nM FIG. 1. Detection of a binary complex (0) of (6R)-5,10-

[14Clmethylene-H4PteGlu4 and human TS by equilibrium dialysis and detection of a ternary complex with d m (0) by ultrafiltration. Inset, the binding of 5,10-[14C]methylene-H4PteGlu4 to human TS in a ternary complex with dUMP analyzed by Scott plot (37).

cofactor in a binary complex, thus the Kd and the stoichiometry of binding in the binary complex could not be determined ac- curately. In contrast, TS bound more 5,lO-methylene- H4PteGlu4 in a ternary complex with dUMP as determined by ultrafiltration. These data were further analyzed by a Scott plot (Fig. 1, inset). It was calculated that only 0.62 mol of 5,lO-methylene-H4PteGlu4 was bound per mol of extensively dialyzed, folate-free dimeric enzyme with an apparent Kd of 720 nM. The stoichiometry of the folate cofactor in the ternary complex (n = 0.62) is significantly less than the stoichiometry of either FdUMP with 5,lO-methylene-H4PteGlu4 (n = 1.7) or that of dUMP with the folate analogs (Table I, n = 1.25-1.6). We considered the possibility that the two sites of human TS bind the folate cofactor with significantly different Kd values and that the binding of the substrate to the second subunit is much weaker. We attempted to measure the Kd for this putative weaker binding site by using an enzyme concentration of 10 p and 5,10-[14Clmethylene-H4PteGlu4 concentrations ranging up to 15 p. We did not observe another Kd value over the range of enzyme and substrate concentrations used in these experiments (data not shown).

Evidence for the Binding of D1694G1u4 to the T h o Sites of Human TS with Substantially Different Binding Affinities- The binding of [14ClD1694Glu4, a close structural analog of the folate cofactor, to human TS (40 nM) in a ternary complex with dUMP was determined by equilibrium dialysis, with inhibitor concentrations ranging up to 400 nM. The results are presented in Fig. 2. The function that fitted the data was not linear (Fig. 21, indicating the presence of two binding sites with different binding affinities. Using the segregation procedure outlined by Pattishall et al. (341, Kd values of 1.1 and 28 nM for the two binding sites were estimated.

Effect of the Folate Cofactor on the Binding of FDDF- Formation of the binary or ternary complex of L3H1FDDFGlul with 5.0 p enzyme, dUMP, and up to a 20 concentration of the inhibitor yielded no detectable binding in the binary complex but resulted in binding with an apparent Kd value of 2300 nM and n = 1.6 for the ternary complex (Table 11). We added 1.0 mM 5,lO-methylene-H4PteGlul, a concentration about 50 times

0 50 100 150 Free [I], nM

200

FIG. 2. Binding of D1694Glu4 to human TS in a ternary complex with d m . The line drawn is that of the best fit for two sites with n, = 0.81 and Kdl = 1.1 nM, n2 = 0.85 and Kdz = 28 nM.

more than its K, value, to the equilibrium dialysis buffer. During the equilibration period less than 15% of the substrate was converted to the products. Based on the K , value of the substrate (12 p) and the Kd value of the inhibitor (2.3 p, Table 111, we estimated that 1.0 mM 5,lO-methylene-H4PteG1ul will completely prevent the binding of FDDF to TS over the range of inhibitor used during equilibrium dialysis. Surpris- ingly, the addition of the folate cofactor had no effect on the Kd value of FDDF in a ternary complex, but it reduced the n value by approximately 2-fold (from n = 1.6 to n = 0.9, Table 11). These data are consistent with the possibility that the two active sites of TS are asymmetric and that the folate substrate binds to one site tighter than the other site.

Binding of Either 28431389 or PDDF to TS-Measurements of binding of radiolabeled 1843U89, or PDDF in different enzyme complexes were determined, and data are presented in Table 11. [3H11843U89 (10-100, m) bound TS (160 m) in a binary complex with an apparent Kd of 6 nM and n = 0.5 as determined by equilibrium dialysis. In contrast, the formation of a binary complex of [14C]PDDFGlul (100-2,000 m) and human TS (200 m) by equilibrium dialysis was not detected. Higher concentrations of TS (10 p) and [14ClPDDFGlul (1-50 p) did form complexes, but a high n value (n >lo, data not shown) suggested that PDDF was binding nonspecifically.

Ternary complexes were observed with TSdUMP and either 1843U89 ( K d = 0.1 m, gel permeation) or PDDF (Kd = 46 m, equilibrium dialysis) with a stoichiometry of 0.78 and 1.2 mol of inhibitor per mol of dimeric enzyme, respectively (Table 11). The stoichiometry of binding of either compound was not significantly different than 1.0 but was less than a value of 1.6 observed for FDDF (Table 11). Moreover, in contrast with the results obtained with FDDF, the addition of 1.0 mM 5,lO-methylene-H4PteGlu had no significant effect on the stoichiometry and the binding affinity of either 1843U89 or PDDF in the ternary complexes (Table 11). The addition of 0.2 mM nonradioactive FDDF to the equilibrium dialysis buffer prevented the binding of the radiolabeled PDDF or 1843U89 to TS in a ternary complex with dUMP (data not shown). The data are consistent with the hypothesis that 1843U89 and PDDF bind to the low folate affinity site of human TS with Kd values of 0.1 and 46 nM, respectively, and that the Kd values of 1843U89 or

Folate Analog Binding to Thymidylate Synthase TABLE I1

Dissociation constants and no. of binding sites for monoglutamylated folate analog inhibitors of TS with different additions to the equilibrium mix

All except the ternary complexes of TS.1843U89.dUMP (gel permeation chromatography) were observed by equilibrium dialysis.

1877

Additions FDDF 1S43U89 PDDF

K , n Kd n Kd n

nM nM nM

None NDa 6 ( + U b 0.5 (kO.1) ND dUMP (0.5 m) 2,300 (t720) 1.6 (k0.2) 0.10 (20.06) 0.78 (k0.2) 46 (29) 1.2 (k0.2) dUMP plus (6R,6S)-5,10-methylene-H4PteG1u, (1 mM) 1,800 (2430) 0.9 (k0.02) 0.13 (?0.07) 0.65 (k0.15) 48 (25) 1.1 (kO.1)

Not detectable. Kd values of FDDF and PDDF in a binary complex with human TS were higher than 0.5 and 5 PM, respectively. S.E.

PDDF for the second site were not observed over the range of enzyme and substrate concentrations used in these experiments (Table 11). To detect the K d values for the second site, higher concentrations of the ligands were required. However, because of nonspecific binding of these compounds ( n > 10) at the higher concentrations of enzyme and ligands, we were un- able to determine the Kd values for the second site by equilibrium dialysis methods directly (data not shown).

Instead, we measured the Kd values for 1843U89 or PDDF for the second site by competition experiments. Ternary complexes of 5,10-[’4Clmethylene-H~PteGlu4~human TS.dUMP were formed and isolated by ultrafiltration in the presence of different concentrations of nonradioactive 1843U89 or PDDF (Fig. 3). Concentrations of the competing ligands which reduced the ternary complex formation by 50% were estimated and fitted to Equation 2 to estimate the Kd values for the second site. The Kd values for 1843U89 or PDDF for the second or the high substrate affinity site were 470 and 353 nM, respectively.

The ternary complex of 5,10-[14Clmethylene-H4PteGlu4~ human TS.dUMP was unstable, and also 5,10-[’4C]methylene- H4F’teGlu4 bound to the ultrafilters nonspecifically (-15%). Thus, we also estimated the Kd values for the second site by forming a ternary complex of [3HlFDDF (6 p ~ ) , human TS (5 p ~ ) , and dUMP (0.5 mM) by equilibrium dialysis in the presence of different concentrations of nonradioactive PDDF or 1843U89. The K d values for PDDF or 1843U89 for the second site were within 30% of the values obtained in the substrate competition experiments discussed above (data not shown). To- gether, the data from equilibrium dialysis and competition experiments suggest that 1843U89 or PDDF bind the two sites of human TS with different Kd values. Monoglutamylated forms of 1843U89 and PDDF bind the low affinity substrate site approximately 5,000 and 9 times more tightly than the second or the high affinity substrate site, respectively (Table I1 and Fig. 3). We also compared the binding of [l4C1PDDF to human or E. coli TS in a ternary complex with dUMP by equilibrium dialysis and observed no differences in the binding of PDDF to human and bacterial thymidylate synthases (data not shown).

Binding of Polyglutamylated Forms of Folate Analogs to TS-Unlike Dl694 and PDDF, diglutamates of 1843U89 accu- mulated as the major species in cells treated with 1843U89 (7). Table I11 shows the results of measurements of binding radiolabeled 1843U89Glu2, PDDFGlu,, or D1694Glu4 to TS in the binary complex or in the ternary complex with dUMP. The binding properties of [3H]1843U89Glu2 to human TS were very similar to those of the monoglutamylated form. Conditions used to study the formation of a binary complex of [3H11843U89Glu2.human TS or a ternary complex of [3H11843U89Gluz.human TSdUMP were identical to those used for 1843U89. h apparent K d value of 10 nM and n = 0.7 in the binary complex and an apparent Kd of 0.37 nM and n = 0.6 for the ternary complex were obtained (Table 111).

The binding of [14C]PDDFGlu4 to human TS in a binary (E,

0 A/-

- PDDF &= 353 f 78 nM

Unlabeled Competing Ligand, nM

FIG. 3. Measurement of the & values for 1843U89 (O), PDDF (A), or 1843U89Glu2 (0) for the second site by competitive binding assays. Ternary complexes of 3 p~ 5,10-[14Clmethylene-H4PteGlu4, 5 p~ human TS, and 0.5 mM dUMP were formed in the presence of different concentrations of nonradioactive 1843U89, PDDF, or 1843U89GluZ and isolated by ultrafiltration. Concentrations of the competing ligands which reduced the ternary complex formation by 50% were estimated and fitted to Equation 2 to estimate the Kd values for the second site. The& values for 1843U89, PDDF, and1843U89Gluz for the second site were 470, 353, and 375 nM, respectively.

Binding of polyglutamylated folate analog inhibitors to human TS in TABLE I11

the binary and ternary complexes

Compound [E.II binary complex [E.I.dUMPl ternary complex

Kd n K d n

nM nM

1843U89Gluz 10 (22)” 0.7 (t0.1) 0.37 ( 4 . 2 ) 0.6 (k0.3)

a S.E.

cedures”).

~~

‘ b o Kd values were estimated from Fig. 2 (see “Experimental Pro-

= 160 n~) and the ternary complex (E, = 40 n ~ ) with dUMP resulted in K d values of 44 and 1.5 nu, respectively (Table 111). The mol of binding sites for PDDFGlu, in the binary or the ternary complex were approximately 2.0. These data are consistent with the conclusion that [14ClPDDFGlu4 binds to both the active sites of the TS dimer with equal affinities. Formation of the [14ClD1694Glu4.human TS binary complex was measured using an enzyme concentration of 160 nM, giving an ap-


P P - FOOF

7.5

6.0

Lo J 3 4.5

2- > 0 ._ w - 2 3.0 0 2

1.5

I--" POOFGLUs

1

f -

* f f

0.0 I I I

0 30 60 90

f / 1843u89GLu2

(6R)-5,10-Methylene H4PteGlu3, @M

FIG. 4. Panel A, inhibition kinetics of human TS by FDDF (O), PDDF (m), and 1843U89 (0) as a function of 5,10-methylene-H4PteG1ul. Inhi- bition constants (K,) of each inhibitor at a specific concentration of 5,lO-methylene-H4PteGlul were estimated. Inhibition constants were normalized with the K, = 1.0 for each inhibitor determined at the lowest concentration (0.25 p) of the substrate used. Panel B, inhibition kinetics of human TS by PDDFGlu, (m) and 1843U89Glu2 (0) as a function of 5,10-methylene-H4PteGlu,.

parent Kd value of 336 nM and n = 1.6 (Table 111). The stoichiometry and the two different Kd values for D1694Glh in a ternary complex with dUMP were obtained from Fig. 2 and are presented in Table I11 for comparison purposes.

A second Kd value of 1843U89Glu2 in a ternary complex with dUMP determined by substrate competition experiments (Fig. 3) was 375 m. Thus, like the monoglutamate, the digluatamate of 1843U89 binds the active sites of TS with unequal affinities.

Znhibition of TS by Folate Analogs-Values of Ki for monoglutamates of 1843U89, PDDF, and FDDF were determined over the full range of 5,lO-methylene-H4PteGlul for which re- liable measurements could be made (Fig. 4A). As expected for a noncompetitive inhibitor, the inhibition of human TS by either 1843U89 or PDDF was independent of substrate up to a concentration of 200 p ~ , a value that is about 20 times higher than the K, for 5,10-methylene-H4PteGlul. However, when substrate concentration ranged from 200 to 500 p ~ , there was a decrease in inhibition, and Ki values for 1843U89 and PDDF

A. Binary Complex

TS dUMP PHjdUMP

1 1

B. 9 TS dUMP PDDF

C. TS - dUMP 1843U89

12 16 20 Fraction Numbers

FIG. 5. Gel permeation liquid chromatography (GLPC) of non-

and free 6-[3HldUMP during GPLC are indicated by arrows in panel A. productive complexes of human TS. Retention times of human TS

Elution of radioactivity during GPLC of reactions prepared with 6-[3HldUMP (1.4 p ~ ) and human TS (30 m) (panel A); 6-L3HldUMP, human TS, and nonradioactive PDDF (50 p) (panel B); and 6-L3H1dUMP, human TS, and nonradioactive 1843U89 (10 p ~ ) (panel C ) . Reactions were incubated for 16 h at 25 "C prior to GPLC. Aliquots (0.05 ml) of the reactions were applied to a TSK G3000 and eluted at 1.0 mumin; 1.0-ml fractions were collected and counted for radioactivity.

increased by about 2-fold (Fig. 4A). By comparison, inhibition of human TS by FDDF was more responsive to the increase in the concentration of 5,lO-methylene-H4PteGlul (Fig. 4A). As much as a 12-fold increase in Ki values for FDDF was observed (Fig. 4A). Because FDDF is not a tight binding inhibitor, we also analyzed the data from Fig. 4A by using the Michaelis- Menten equation (data not shown). The analyses revealed that FDDF had effects on both the K,,, for 5,lO-methylene- H4PteGlul and the V,, values, which is a characteristic of mixed noncompetitive inhibition. For example, in the presence of 37 1.1~ FDDF the K, for 5,lO-methylene-H4PteGlul increased by 7-fold, and the V,, value decreased by a factor of 2.5. Replots of primary reciprocal plot data gave values of Kii (intercept) and Ki, (slope) of 1.5 and 30 p ~ , respectively.

Values of Ki for PDDFGlu, also increased with an increasing concentration of 5,lO-methylene-H4PteGlu3 as a substrate (Fig. a), suggesting that PDDFGlu5 is also a mixed noncompetitive inhibitor of TS. However, values of Ki for the diglutamylated 1843U89 remained invariant over the range of the triglutam- ylated substrate examined (Fig. a), consistent with noncompetitive inhibition.

Lack of Correlation between Formation of Nonproductive Complexes and Mode of Znhibition of TS by a Folate Analog- Previously it was reported (17) that after formation of a reversible L. casei TS.PDDF.dUMP complex, a slow isomerization occurred which resulted in the formation of a stable and nonproductive ternary complex. The formation of this complex was implicated in the noncompetitive kinetics for PDDF. We compared the ability of two noncompetitive inhibitors, PDDF and 1843U89, and a mixed type inhibitor, FDDF, to form a stable ternary complex with [3HldUMP and human TS. Therefore, the preformed binary complex of human TS.[3HldUMP (Fig. 5 A ) and ternary complexes of TS.[3HldUMP.PDDF (Fig. 5B), TS.[3H]dUMP.FDDF (data not shown), and TS.[3HldUMP.

Folate Analog Binding to Thymidylate Synthase 1879

I I I I

15 30 45 Free [I], nM

hc. 6. Binding of [“C]1843U89 to human TS with nonradioactive 0.6 m dUMP (A), 0.5 m dTMP (O), dUMP, and 1.0 mrd (6R,6S)-5,10 methylene-H.,PteGlu, (A), dTMP and 0.5 m H,PteGlu (O), and without additions (e).

1843U89 (Fig. 5C) were passed through a gel filtration column to separate the enzyme bound from free 6-L3H]dUMP. A fraction of the 6-L3H1dUMP eluted with the enzyme peak as a binary complex (Fig. 5 A ) or a ternary complex with PDDF (Fig. 5B) , clearly separate from the nucleotide, and was TS-bound as judged by nitrocellulose binding assay (17). In contrast, the results obtained with another noncompetitive inhibitor, 1843U89, were very different. Minimal L3H]dUMP was associ- ated with human TS in the presence of 10 1.1~ 1843U89 after 16 h of incubation (Fig. 5C). The formation of the stable binary complex of human TS and i3H1dUMP was inhibited by 10 w 1843U89.

The formation of binary TS.[3HldUMP and ternary complexes of TS.[3H]dUMP-PDDF and TS.[3HldUMP.FDDF were time- and concentration-dependent (data not shown). The rates of formation of these three complexes were identical. However, the rates of dissociation of 6-13H]dUMP from the binary TS.[3HldUMP and ternary complexes of TS.[3H]dUMP.PDDF and TS.[3HldUMP.FDDF (kOtf, h-’) were 0.86,0.32, and 0.026, respectively. The data suggest that both PDDF, a noncompetitive inhibitor, and FDDF, a mixed type inhibitor, can stabilize the human TS.[3HldUMP complex by decreasing its rate of dissociation.

Noncompetitive Inhibitors Are Not Product Analogs-Santi and Danenberg (2) have suggested that noncompetitive inhibitors are the analogs of HzPteGlu, one of the products of the TS reaction. To explore this possibility further we studied the formation of complexes of a noncompetitive inhibitor [3H]1843U89 (10-100 m) with the enzyme (200 m) by equilibrium dialysis in the presence of the products and substrates of the TS reaction. The results are shown in Fig. 6. Based on the Ki values of the products and the Kd value of 1843U89 (Table 11), we hy- pothesized that if 1843U89 was a product analog, then dTMP (500 w) alone or dTMP plus HzPteGlul (500 w) will completely prevent the binding of 1843U89 to TS over the range of

the radiolabeled inhibitor used during equilibrium dialysis (Fig. 6). On the contrary, significantly more 1843U89 bound TS in the presence of dTMP, or dTMP plus HzPteGlul (Fig. 6). Moreover, as expected from its noncompetitive mode of inhibition, the addition of (6R,6S)-5,10-methylene-H4PteGlu1 to the equilibrium reaction had no effect on the formation of a TS.[3H]1843U89.dUMP complex. Similar results were also observed with PDDF (data not shown).

DISCUSSION

The results obtained from the measurement of binding of folate substrates and analogs to dimeric TS suggest that the two substrate binding sites exhibit ligand-induced negative cooperativity. For example, only 0.6 mol of 5,lO-methylene- H4PteGlu4 bound per mol of dimeric enzyme in a ternary complex (Fig. 1) rather than the 2 mol expected if both subunit sites were available for binding. Several nonspecific factors such as heterogeneity of the enzyme and the ligands and presence of inhibitors can give rise to negative deviations from the simple binding process. The following evidence rules out these factors as the cause of the observed half-site reactivity of TS. The reduced binding sites were not caused by enzyme heterogeneity since the number of active sites per dimer was 1.7 (-85% active TS) as determined by titration of TS and 5,lO-methylene- H4PteGlu4 with FdUMP. This is further confirmed by the binding of about 1.6 mol of [3H]dUMP in a ternary complex with folate analogs (Table I), 1.6 mol of L3H1FDDF in a ternary complex with dUMP (Table 111, or 1.7 mol of [l4CIPDDFGlu4 in a binary or a ternary complex with dUMP (Table 111). Human TS purified after the affinity column was bound to a small DEAE-cellulose column and washed extensively, eluted with high salt in buffer, and then dialyzed extensively; this should have removed any residual folates. The observation that TS bound 1.6 mol of [3H]FDDF or 1.7 mol of [l4C1PDDFGlu4 also suggests that the enzyme is folate-free. The lower values for stoichiometry in the binding assays are also not caused by impure preparations of the folate substrate. The folate substrate and the analogs used in this study were better than 97% pure, and the concentration of each ligand was checked by two independent methods. Thus we conclude that asymmetric binding of the folate substrate and analogs is the major factor that determines the inhibition kinetics of TS by a particular folate analog.

Active site-site interaction and the asymmetry of the two sites for this enzyme have also been suggested based upon experiments performed with the nucleoside substrate, dUMP, and its analogs. Aull et al. (46) suggested that the two sites of L. casei TS are nonequivalent because FdUMP decayed more rapidly from one site when in a ternary complex with 5,lO- methylene-H4PteGlu. Many laboratories (47-52) have presented data that support the hypothesis that only one binding site for dUMP is accessible on the free enzyme in the absence of folate cofactor. The subsequent binding of the cofactor allows binding of dUMP at the second site (47-53). Other groups have published data supporting asymmetrical active sites. Beau- dette et al. (54) and Lockshin and Danenberg (55) demon- strated that the two active sites in L. casei TS bind dUMP with different affinities both in binary and ternary complexes. Garvey and Santi (35) showed that Leishmania major TS binds dUMP to one subunit which slowly converts to a catalytically incompetent form that does not inhibit the activity of the other subunit. Aull et al. (56) observed that removal of a carboxyl- terminal valine from one subunit by carboxypeptidase A results in the inactivation of both the sites of L. casei TS. Finally x-ray crystallography has shown that each substrate binding site of TS is composed of amino acid residues from both subunits (3-5).


TABLE IV Summary Of Kd and K, values and mode of inhibition for various folate analog inhibitors of TS

Ternary complex with dUMP Kda Site A Site B Kd

nM nht

Inhibitor Inhibition constant Mode of

inhibition

1843U89 1843U89Glu2 PDDF 350 (280) 46 (29) PDDFGlu, 1.5 (20.2) FDDF 2,300 (2720) 1,800 (2430) 1,500 (2300) D1694Glu4 Substrate = 5,10-methylene-H4PteGlu, 720 (t110) NDd K,,, = 1,900 (t400) Substrate

470 (t120)b 0.10 (20.06) 0.09 (20.06) Noncompetitive 375 (282) Noncompetitive

7.0 (t2) Noncompetitive 1.2 (e0.3) Mixed

Mixed 1.1 (20.3) 28 (25) 1.0' Mixed

0.37 (20.2) 0.13 (e0.05)

1.5 (20.2)

Kd values for site A for 1843U89, 1843U89Glu2, and PDDF were obtained by competition experiments (Fig. 3) and for FDDF from Table 11. Kd values for the two sites for PDDF Glu4 (Table 111) and D1694Glu4 (Fig. 2) were observed directly by equilibrium dialysis. Kd value of 28 nM for D1694Glu4 was assigned to site B based on competition experiments with 1843U89 (data not shown).

S.E. From Ref. 18. Not determined.

We have observed five different examples of subunit asymmetry in TS which are summarized in Table IV. The results in Fig. 1 suggested that 5,lO-methylene-H4PteGlu4 bound only one of the subunits of dimeric TS with a Kd of 720 nM in a ternary complex. These results are in apparent contradiction with those obtained by others by equilibrium dialysis (57), crystallographic (11) and other studies (1-2). These workers showed that in the presence of FdUMP, mono or tetraglutamylated folate substrate occupied both the active sites of TS as covalent complexes. However, they did not determine dissociation constants for the folate substrate for each site. One of the possible explanations for the difference could be that the binding of the substrate to the second subunit is much weaker, and the Kd for this binding could not be determined by ultrafiltration over the range of enzyme and substrate concentrations used in these experiments (Fig. 1). Two observations support the hypothesis that human TS has two binding sites for the folate cofactor with appreciably different Kd values. First, the addition of the folate cofactor decreased the number of binding sites for FDDF/mol of TS only by a factor of 2 (Table 111, im- plying the presence of a weak and a tight binding site for the folate substrate. Second, binding of compounds that are close structural analogs of folate occurred with substantially different binding affinities depending on the analog (Fig. 2 and Table IV).

The inhibition kinetics of TS by a folate analog can be readily explained by its binding to TS in relation with the substrate (Table IV). For discussion purposes, the site to which the folate substrate binds more tightly is designated site A, and the second site is designated site B. Based on mode of TS inhibition the folate analog inhibitors used in this study may be divided into two classes (Fig. 4, A and B and Table IV). The noncompetitive inhibitors 1843U89, PDDF, and 1843U89Glu2 belong to the group of folate analogs which bound site B severalfold more tightly than site A (Table IV). The second class of mixed inhibitors (FDDF, PDDFGlu4, and D1694Glu4; Fig. 4, A and B , Ref. 19, and Table IV) bourid both sites equally well (FDDF and PDDFGlu,) or site A more tightly than site B (D1694Glu4). Because of the tighter binding to site A (substrate binding site) these folate analogs competed with the substrate for binding which is reflected by a decrease in inhibition of TS with an increase in the concentration of the substrate (Fig. 4, A and B, and Ref. 19). The analogs that belonged to the second class, however, also bound to site B with about equal affinity thus resulting in a decrease in V,, with the increase in the concentration of the inhibitor (data not shown; 19).

We conclude that the asymmetric binding of folate analogs is the major factor that determines the inhibition kinetics of TS

by a folate analog. However, it has been suggested by others (2, 17) that the formation of stable dead-end binary complexes of the folate analogs with free enzyme and/or ternary complexes with dUMP could lead to noncompetitive kinetics. No correlation between the mode of TS inhibition and the formation of the nonproductive ternary complexes was observed (Fig. 5). Both a noncompetitive (PDDF, Fig. 5B) and a mixed type inhibitor (FDDF, data not shown) stabilized the nonproductive TS.[3HldUMP complex by decreasing its rate of dissociation. In contrast, another noncompetitive inhibitor 1843U89 inhibited the formation of the stable binary complex (Fig. 50. Moreover, the formation of these nonproductive complexes is very slow and influences the mode of inhibition only during longer incubation periods (17). During this study only the initial rates of reaction were considered.

The evidence presented in this paper suggests that the formation of the stable binary and/or ternary complexes of TS have a minimal role in determining inhibition kinetics of TS by a folate analog. No stable dead-end binary complexes of the folate analogs with free enzyme were detected by gel permeation chromatography (data not shown). However, weak binary complexes of some of the folate analogs with TS were observed by equilibrium dialysis (Tables I1 and 111). These binary complexes were readily converted to tighter binding ternary complexes by the addition of dUMP (Tables I1 and 111). A possible mechanism based on crystallographic data, by which a binary complex of the folate and TS can change into a productive ternary complex with dUMP, was suggested by Matthews et al. (5). Recently Kamb et al. (6) have solved structures of two binary complexes of TS with either mono or polyglutamylated PDDF. These structures suggest that the dUMP binding site is accessible in the binary complex, and in the presence of dUMP a binary complex of TS and the folate can undergo a conforma- tional transition to the catalytically competent structure.

Direct involvement of subunit interaction and negative cooperativity in catalysis has been well documented with several enzyme systems (58). Apparently many enzymes have evolved to accomplish two thermodynamically conflicting roles; 1) se- questering the substrate by binding tightly, and 2) providing a weaker destabilized mode of binding to promote catalysis (59). The flip-flop (60) and alternate-site (61) models explain the possible mechanisms by which dimeric and polymeric enzymes can facilitate the reaction by alternating catalytic sites between two subunits. The asymmetric half-site reactions involving the folate substrates of TS suggest that maybe an alternating site cooperativity mechanism is at work in this enzyme. According to this model, TS functions asymmetrically so that binding reactions on one subunit are coupled to catalytic conversions on

Folate Analog Binding to Thymidylate Synthase 1881

the other subunit, and after completion of a full catalytic cycle, the subunits reverse roles. In Table IV, site A would be the site designated for substrate binding, and site B would be on the catalytic subunit. A similar “ligand-induced sequential model” for TS catalysis was also proposed by Danenberg and Danen- berg (50). According to this model, cooperative effects would also result from different binding parameters in the two sites.

Crystallographic studies suggest that the two active sites of unliganded TS are symmetrical (3-5). The “half-of-the-sites effect” therefore is induced by a change in conformation of TS by the initial binding of dUMP followed by even larger changes caused by the binding of the folates (1-5) and not by preexisting asymmetries. Many other enzymes also appear to operate by the ligand-induced negative cooperativity mechanism (58). The tetraglutamate of Dl694 binds both of the sites in a binary complex with equal affinities. The binding of dUMP apparently induces an asymmetry in the TS subunits, resulting in one weaker and one tighter binding site for this compound (Table I11 and Fig. 2). The results, however, do not allow us to exclude all types of preexisting asymmetry, since only one tighter binding site for dUMP (Table I and Refs. 47-56) or for mono or diglutamylated 1843U89 (Tables I1 and 111) is accessible on the free enzyme. Recent x-ray crystallographic data also showed that the binding of PDDFGlu4 in the two active sites is not symmetric in that the quinazoline ring is less ordered and is rotated further into the dUMP binding site in the second monomer than in the first (6).

Formation of the ternary complexes of the folate substrate or the analogs with dUMP and TS displayed synergism since these compounds bound human TS more tightly in a ternary than in a binary complex. These results are consistent with the ordered mechanism of substrate binding and product release for TS (13-14,57,62). However, the extent of synergism varied for each compound. For example, PDDFGlu4 bound human TS in a ternary complex only 10 times more tightly than in a binary complex, as compared with a difference of several thou- sand-fold for PDDF (Tables I1 and 111). Based on product inhibition studies Lu et al. (63) proposed that the order of tetraglutamylated substrate binding and product release for pig liver TS may be reversed when compared with the monoglutamylated substrate. Ghose et al. (64), however, showed that this difference was only observed in phosphate buffer but not in Tris chloride. We confirmed the observations of Radparvar et al. (65) and Ghose et al. (64) and found no appreciable difference in product inhibition between mono and polyglutamylated substrate in Tris chloride buffer in E. coli and human TS (data not shown). The data are consistent with the hypothesis that the binding of dUMP precedes that of the mono and polyglutamylated folate substrate, and the release of dTMP follows the release of HzPteGlu, (62). The observation that tetraglutamylated substrate bound human TS in a ternary complex about 30 times tighter than in the binary complex (Fig. 1) is also in agreement with this proposal.

The results presented in this paper show that several folate analogs and the substrate 5,lO-methylene-H,PteGlu4 bound the two sites of human TS with different affinities. The mode of inhibition kinetics by the folate analogs of TS is readily explained by their binding in relation with the folate substrate.

the chemical synthesis of Dl694 and BW1843U89 and G. Nair for Acknowledgments-We thank S. Dickerson and W. Pendergast for

supplying PDDF. We acknowledge the technical assistance of R. Riggs- bee and thank J. Burchall for encouragement and support.

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