ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 260, No. 1, January, pp. 342-350,1988
Characteristics of Thymidylate Synthase Purified from a Human Colon Adenocarcinoma’y2 SAEED RADPARVAR, PETER J. HOUGHTON,
JANET A. HOUGHTON3
Laboratories fbr Developmental Therapeutics, L?ivisimz of Biochemical and Clinical Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38101 Received May ‘7, 1987, and in revised form September
Thymidylate synthase has been purified >4000-fold from a human colon adenocarcinoma maintained as a xenograft in immune-deprived mice. In this disease, the enzyme is an important target for the cytotoxic action of 5-fluorouracil, which is influenced by the reduced folate substrate CHs-H4PteGlu. Due to the importance of this interaction, and the existence in cells of folate species as polyglutamyl forms, the interaction of folylpolyglutamates with thymidylate synthase was examined. Polyglutamates of PteGlu were used as inhibitors, and the interaction of CHz-H4PteGlu polyglutamates as substrates or in an inhibitory ternary complex were also examined. Using PteGlulm7, Ki values were determined. A maximal 125-fold decrease in Ki was observed between PteGlul and PteGlu4; further addition of up to three glutamyl residues did not result in an additional decrease in Ki. Despite the increased binding affinity of folylpolyglutamates for this enzyme, no change in the Km values for either dUMP (3.6 PM) or CHs-H4PteGlu (4.3 PM) were detected when polyglutamates of [6R]CHz-HIPteGlu were used as substrates. Product inhibition studies demonstrated competitive inhibition between dTMP and dUMP in the presence of CH2-H4PteGluS. In addition, CHZH4PteG1u4 stabilized an inhibitory ternary complex formed between FdUMP, thymidylate synthase, and CHs-H4PteGlu4. Thus the data do not support a change in the order of substrate binding and product release upon polyglutamylation of CHz-H4PteGlu reported for non-human mammalian enzyme. This is the first study to characterize kinetically thymidylate synthase from a human colon adenocarcinoma. o 19% Academic press, I,,~.
Thymidylate synthase (EC 22.214.171.124) catalyzes the conversion of deoxyuridylate (dUMP)4 to thymidylate (dTMP), involvi Supported by NC1 Award CA 32613, and by the American Lebanese Syrian Associated Charities. ’ The Materials and Methods section of this paper appears as a Miniprint Supplement. ’ To whom correspondence should be addressed. 4 Abbreviations used: dUMP, deoxyuridylate; dTMP, thymidylate; [GRS]CHa-HIPteGlu, a racemic mixture of the natural [6R] and unnatural [6s] diastereoisomers of 5,10-methylenetetrahydrofolate; H*PteGlu, dihydrofolate; PteGlu, folk acid; FUra, 5fluorouracil; FdUMP, 5-fluorodeoxyuridylate; TMDGT buffer, 0.1 M Tris-HCl, pH 7.5, containing 1.5 mM MgCla, 2 mM DTT, 10% glycerol, and 0.1% Triton X-100; PCA, perchloric acid; DTT, dithiothreito1. 0003-9861/88 $3.00 Copyright All rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
ing the transfer and reduction of a methylene group from 5,10-methylenetetrahydrofolate (CHe-H4PteGlu) to dUMP, with simultaneous oxidation of the folate cofactor to dihydrofolate (HsPteGlu (1, 2)). This constitutes a key enzyme in pyrimidine biosynthesis, providing the only source of dTMP synthesized de novo in mammalian cells. It also forms an important target for the antineoplastic agent 5fluorouracil (FUra (3-5)). In colon adenocarcinoma, a disease that is generally refractory to treatment with chemotherapeutic agents, FUra has proven to be one of the most effective single agents, inducing response rates in approximately 20% of patients (6, 7). Data derived in these laboratories using human colon ade-
nocarcinoma xenografts have correlated tumor sensitivity to FUra with both the level and duration of inhibition of thymidylate synthase (8). 5-Fluorouracil, after metabolism to 5-fluorodeoxyuridylate (FdUMP), forms a covalent ternary complex with thymidylate synthase and CHz-H4PteGlu that is inhibitory to enzyme activity (4, 9,10). The importance of the concentration of CHz-H,PteGlu in forming and maintaining a stable covalent complex is well established (8, 9, 11). Studies from many laboratories have demonstrated that intracellular folate derivatives are present mainly as polyglutamy1 derivatives containing from 2 to 10 glutamyl residues linked through their y-carboxyl groups (12-17). Polyglutamates generally have a higher affinity for folate-dependent enzymes in comparison to the corresponding monoglutamates (18-27), and may have a regulatory function in folate metabolism via effects upon Km, vtnax, or Ki values (18, 21, 24-33). The effect of polyglutamylation of CHz-HIPteGlu on the reaction kinetics for mammalian thymidylate synthase appear variable, and dependent upon species (25, 26,30,33). Consequently, either a decrease (30, 33) or no change (25, 26) in Km values for CHz-HIPteGlu have been observed with increasing polyglutamate chain length. Due to the importance of thymidylate synthase as a target for FUra in human colon adenocarcinoma, and the absence of data that characterize the interaction of folylpolyglutamates with enzyme from
Original supernatant 30-70% (NH&SOs, Al&Gel blue lo-Formylfolic acid
396 212 192 186b
a One unit represents the metabolism *The concentration of [6-aH]FdUMP assay.
Protein (w) 4631 2135 40 0.49
Purification of thymidylate synthase was achieved from human colon adenocarcinema xenografts, despite low concentration of enzyme. Details of the yields, specific activity, and total units obtained for a representative purification are presented in Table I. Since these solid tumors contain necrotic central cores, the ammonium sulfate fractionation step was included even though the extent of purification (1.2-fold) was minimal. Subsequent elution from a column of Affi-Gel blue (Fig. 1) resulted in a further 47-fold purification of the enzyme. The principal step in the purification procedure, however, was the use of the affinity column of lo-formylfolic acid, where the highest degree of purification (a further 79-fold) was obtained. Overall, the three steps resulted in a 4401-fold purification of thymidylate synthase, and the enzyme was used without further purification or concentration for kinetic studies. Four separate thymidylate synthase preparations were used for these assays. The purification procedure was reproducible, the average-fold purification being 3916 + 399 (from four purifications). Percentage recovery in different preparations ranged from 29 to 78%.
PURIFICATION OF THYMIDYLATE SYNTHASE FROM HxVR&
this source, the current study was conducted. A human colon adenocarcinoma maintained as a xenograft line in immune-deprived mice was used as the constant source of solid tumor tissue.
I HUMAN COLON ADENOCARCINOMA XENOGRAFTS
Volume (ml) 180 70 8 7
sp act (unit/mg) 0.086 0.099 4.8 378.5
of 1 nmol dUMP/h to dTMP. binding sites was 4.8 nM, determined
100 54 48 47
1 1.2 55.8 4401
using a charcoal
FIG. 1. (A) Elution of thymidylate synthase from a column of Affi-Gel blue (10 X 2 cm) as described under Materials and Methods. (0) Protein (mg/ml); (0) 3Hz0 released (dpm X 10m5);( * * .) of thymidylate synthase using lo-formylfolic Gradient of 0.2 to 0.6 M KCl. (B) Final purification acid coupled to Sepharose (12 X 1.5 cm) according to the procedure of Banerjee et al. (50). Details of all procedures are provided under Materials and Methods. Symbols are the same as in A.
Inhibition constants (Ki) associated The effect of polyglutamylation of with PteGlu, (n = 1 to 7 glutamyl resi- [GR]CHz-HIPteGlu on kinetic parameters dues) were determined using [6R]CHB- for dUMP were subsequently determined. HIPteGlul as the folate substrate in each case. A representative experiment is shown in Fig. 2 using PteGluG as the inhibitor. Inhibition was competitive between PteGlu, and [GR]CHz-HIPteGlul, irrespective of the polyglutamate chain length of PteGlu. Ki values were illustrated from replots of the data, as shown in the inset to Fig. 2. Under the standard assay conditions, a significant decrease in Ki, representing increased binding affinity was de-0.2 -0.1 0 termined, as the glutamyl residues on PteGlu were increased from 1 to 4 (Fig. 3). Binding affinity subsequently decreased slightly between PteGlu, and PteGlu7. Ki I I I values (PM f 1 SE) for PteGlul to PteGlu7 -.05 -.04 -.03 -.02 -.oi 0 .ot .02 .03 were found to be 7.6 +- 1.3, 1.32 & 0.36, 1.0 + 0.34, 0.06 + 0.02, 0.10 + 0.03, 0.20 + 0.41, [(6R)CH2- H,PteGlu, I-‘(pM)and 0.24 k 0.05, respectively. A large deFIG. 2. Inhibition of thymidylate synthase by crease in Ki was observed between PteGlm plots of initial reaction and PteGluz (5%fold), although the great- PteGluG. Double-reciprocal est change occurred between PteGlus and velocity against [(6I?)CHa-H4PteGlul] in the presence of 10.9 FM [5-3H]dUMP. At each concentration of inPteGlul, where a 16.4-fold decrease in Ki hibitor, the folate substrate was added as the racewas determined. Overall, the maximal mic mixture [GRS]CHa-H4PteGlul at concentrations change between PteGlur and PteGlul was of 18.2, 25, 37 and 62.5 pM. The concentrations of a 125-fold decrease in Ki. Enzyme kinetics PteGlus inhibitor were (A) 0, (0) 0.05, (0) 0.15, and were also reproducible between different (A) 0.2 pM. Details of the assays are presented under batches of enzyme (data not shown). Materials and Methods.
Km AND V,,,
EFFECTOFPOLYGLUTAMYLATIONON FOR [GR]CHa-H,PteGlu
[GR]CH,-H,PteGlu Km (PM f SE)
1 , \,<-: o
No of Glutamyl
FIG. 3. The relationship between inhibition constant (Ki) and polyglutamate chain lengths of PteGlu, for n = 1 to 7.
K, (PM) and relative I’,,, values for dUMP are presented in Table II. No significant change in the K, for dUMP was obtained in this analysis (range 2.6 to 3.9 PM). In addition, V,,, determinations for dUMP were also similar when either short-chainor long-chain-length polyglutamates of CHz-HIPteGlu were used as substrates. Kinetic parameters for the metabolism of [6R]CHz-H4PteGlu, to H,PteGlu, were measured, and are presented in Table III. These results were similar in principle to kinetic data derived for dUMP. Although a significant increase in binding affinity of folylpolyglutamates (as illustrated by PteGlu,) occurred with increasing polyglutamate chain length, this did not cause a similar change in Km or relative I’,,, values for the substrates CHe-HIPteGlu,. Product inhibition studies were subse-
[GR]CHa-HIPteGlu, [GR]CHa-HIPteGlu, [GR]CHa-H,PteGlua [GR]CHa-H,PteGlu, [GR]CH,-H,PteGlus [GR]CHa-H,PteGlus [GR]CHa-H,PteGlu,
4.3 6.5 3.5 10.9 8.7 8.3 4.3
f It f f f f f
0.9 1.0 0.4 3.4 0.7 2.9 0.6
1.00 0.61 0.96 0.99 0.77 0.91 1.04
quently conducted to determine whether the lack of decrease in K, for CHBH,PteGlu upon polyglutamylation could be explained due to a change in the order of substrate binding and product release as suggested by Lu et al. (25) for thymidylate synthase isolated from fetal pig liver. Consequently, the nature of the competition between dUMP and dTMP was determined using [6R]CHz-HdPteGluB as the fixed substrate (Fig. 4). Computer fitting for competitive inhibition yielded a 2-fold smaller variance than for noncompetitive inhibition; variance for the former was 1.36 X lop5 (nmol/min)’ and for the latter, 2.74 X lop5 (nmol/min)‘. Based upon the (x2) test for goodness of fit, the probability
EFFECTOFPOLYGLUTAM~LATIONOF [GR]CH,-H,PteGlu ON KINETIC PARAMETERSFOR dUMP dUMP Substrate [GR]CHa-H,PteGlui [GR]CHa-HIPteGlu, [GR]CH,-H,PteGlua [GR]CH,-H,PteGlul [GR]CHa-HIPteGluS [GR]CH,-H,PteGluG [6R]CHp-HIPteGlu,
K,,, (PM 3.6 2.7 2.6 2.7 2.6 3.9 2.9
f 1.5 f 0.4 + 0.3 ?I 0.4 2~0.3 f 0.2 f 0.6
VInax 1.00 1.19 1.15 1.38 1.16 1.66 1.42
FIG. 4. Competitive inhibition of the binding of dUMP to thymidylate synthase by dTMP in the presence of [GR]CHa-H4PteGlu5 (30 PM). Concentrations of [5-3H]dUMP were 1.7, 3.0, 4.0, 6.0, and 10.0 pM, and of dTMP were (a) 0, (0) 2, (0) 4, (0) 6, and (M) 10 FM. Reactions were conducted at 37°C over 4 min. Mixtures were processed and data were analyzed as described under Materials and Methods.
of obtaining a x2 value of 13.52 was equal nomas are particularly low in activity (8, to 0.92 for 22 df for the competitive model, 34), which may, in part, explain the effiindicative of a good fit to the data. How- cacy of FUra as an inhibitor of thymidyever, for the noncompetitive model, a x2 late synthase in this disease. Previously, value of 27.28 yielded a P value of 0.20, the human enzyme has been purified from indicating a significantly inferior fit to AML cells (30; which is a disease considered insensitive to 5-fluorouracil, and the data. Inhibition was thus determined where thymidylate synthase is not an imto be competitive in nature. Further information on the order portant target for drugs) or cultured of ligand binding in the presence of MCF-7 breast cancer cells (33), which have CH2-H4PteGlu polyglutamates was pro- been cultured for many years. However, due to low inherent levels, thymidylate vided from an examination of the ability synthase from colon adenocarcinomas is a of CH2-HIPteGlu, to stabilize the ternary covalent complex formed between difficult target for investigation. The use FdUMP, thymidylate synthase, and of affinity ligands has resulted in signifiCH2-H4PteGlu4 (Fig. 5). The data demon- cant levels of purification for thymidylate strate the dependence of the stability of synthases (1470- to 6000-fold (25,26,33,35, the complex upon the concentration of 36)). In the present study from a human solid tumor, purification was comparable CH2-H4PteG1u4, stability increasing with and reproducible, resulting on average in a increasing concentrations of the folate. 4000-fold level of purification with good yields. The specific activity of the purified DISCUSSION enzyme (6.3 nmol/min/mg protein) was in The purification of mammalian thymithe same range reported for other human dylate synthases has been difficult due to thymidylate synthases isolated from low levels present in tissues and the rela- MCF-7 breast cancer cells (1.5 nmol/min/ tive lability of the enzyme, resulting in low mg protein (33) and AML cells (>18.1 yields (36, 37). Human colon adenocarci- nmol/min/mg protein (30)). The latter enzyme eluted as a single peak when analyzed by polyacrylamide disc gel electrophoresis, although thymidylate synthase isolated from MCF-7 was not further characterized. The overall yield of enzyme obtained from human colon adenocarcinoma xenografts in the current study was low, consequently, further analysis of pu, \y 1 rity was not performed. LB0 0 15 30 45 The purification of thymidylate synTime (min) thase from human solid tumors has also been prevented, to date, by the lack of FIG. 5. Influence of CH,-HIPteGlul on the stability availability of a constant source of enzyme of the FdUMP-thymidylate synthase-CHz-HIPtefrom the same tissue resulting in the use Glul complex. Ternary complex was formed between [6-3H]FdUMP (100 nM), CHz-HIPteGlul (1.5 PM), and of cultured cell lines (33, 36). The maintethymidylate synthase at 3’7°C over 45 min prior to nance of human colon tumors, established dilution and addition of CHz-HIPteGlul at concendirectly from patient material, as xenotrations of (a) 0.38, (A) 0.88, (0) 1.9, and (A) 2.9 pM. grafts in mice, has made the current study An excess of nonradiolabeled FdUMP (100 pM) was feasible for the first time. added to initiate the exchange with [6-aH]FdUMP. This study comprises the first kinetic Reaction mixtures were incubated at 3YC; at time 0, characterization of thymidylate synthase 15, 30, and 45 min, 807~1 aliquots, in duplicate, were purified from a human solid tumor where removed and processed as described under Materials the enzyme is an important target for the and Methods. Correlation coefficients (?) from lincytotoxic action of 5-fluorouracil in vivo. ear regression analysis were 0.967, 0.963, 0.980, and Estimates of K, for dUMP (1.7 to 9 PM) 0.998, respectively.
characterized using thymidylate synthase from various sources (11, 25, 35, 37-41) have fallen in a narrower range than those determined for the active isomer of CHz-HIPteGlu (15.2 to 105 pM (11, 22, 25, 26, 33, 35, 37, 38, 40-42)). This may reflect the greater difficulty in preparing and handling the latter substrate due to problems associated with its stability, the purity of the enzyme preparation (39), or real differences dependent upon the source of the enzyme. For human colon adenocarcinoma enzyme, the K, value for dUMP with [GR]CHz-H4PteGlu1 as the substrate was 3.6 pM; the Km for [GR]CHz-HIPteGlul was 4.3 PM. Both of these values are at the lower end of the range, and are similar to data obtained for fetal pig liver thymidylate synthase (25). Physiological folates exist in cells as polyglutamate derivatives, which are highly charged species and function as retention forms of these cofactors (27). They also possess enhanced binding affinity for several folate-requiring enzymes, including thymidylate synthase (22, 30, 33), methylenetetrahydrofolate reductase (24), and AICAR transformylase (18), reflected in a decrease in the Ki or I&o values of inhibitors, and enhanced rates of catalytic conversion of the folate substrates, particularly in decreased Km values. For mammalian thymidylate synthases, the degree of change in Ki or IC& values for folylpolyglutamates may depend upon the source of the enzyme and the compound under investigation (33, 43, 44). For non-human mammalian thymidylate synthases purified from calf thymus (26) or fetal pig liver (25), a significant decrease in I& or Ki was observed for polyglutamates of PteGlu (maximally 27- and 175-fold, respectively). However, for the latter enzymes, this increased binding affinity did not translate into a decrease in Km for the natural substrate after polyglutamylation. As polyglutamates of PteGlu had not previously been evaluated as inhibitors of the human enzyme, these were employed in the current study. Our results with thymidylate synthase purified from a human colon adenocarcinoma clearly showed that a significant change in bind-
ing affinity occurred with polyglutamate derivatives of PteGlu, increasing up to the tetraglutamate. These results are similar to those obtained for fetal pig liver enzyme (25). By contrast, with calf thymus thymidylate synthase, the penta- or hexaglutamate of PteGlu demonstrated maximum inhibition (26). A difference in Km has been reported for [6R]CHz-HIPteGlul and the corresponding pentaglutamate using human thymidylate synthases; for the AML enzyme (30), values were 15.5 and 1.1 pM (14-fold), and for enzyme from breast cancer cells (33), 22.6 and 0.63 pM (36-fold), respectively. It is of interest, however, that in our studies, increased binding affinity of folylpolyglutamates was not paralleled by a decrease in the Km values for [6R]CHz-HIPteGlu upon polyglutamate formation. These results are different from data obtained using thymidylate synthase purified from these other human sources (30, 33), but are similar to properties of both calf thymus and fetal pig liver enzymes (25, 26). The interaction of dUMP and [6R]CHzH4PteGlu1 with thymidylate synthase from different sources has been well characterized (9, 11, 25, 35). Binding is mediated by an ordered mechanism, with the nucleotide binding to the enzyme prior to the folate, and H,PteGlu dissociating prior to dTMP, such that inhibition between dTMP and dUMP is competitive in nature. With enzyme isolated from fetal pig liver, Lu et al. (25) suggested that inhibition between dTMP and dUMP became noncompetitive when [6R]CHz-H4PteGlu was polyglutamylated, such that a change in the order of substrate binding and product release could explain the lack of decrease in Km for CHz-HIPteGlu upon polyglutamylation. In the current study, however, inhibition by dTMP of dUMP binding remained competitive in nature in the presence of [GR]CHz-H4PteGlu5, suggesting no change in the order of the reaction for human colon adenocarcinoma enzyme. Further data to support this contention were obtained from a study demonstrating the influence of CHz-H4PteGlu4 on the stability of the FdUMP-thymidylate synthase-CH2-H4PteG1u4 ternary com-
plex. A change in the order of ligand binding would result in the stability of the complex becoming independent of the folate concentration when CH2-H4PteGlu was polyglutamylated. However, in the presence of CH2-H4PteGlu4, the stability of the complex increased with increasing folate concentration. Possible explanations for the different effects of polyglutamylation on Ki for PteGlu, and I& for CH,-H,PteGlu, could include (i) a different mode of binding in the active site cavity, or (ii) that the initial complex is not important for substrate Km. These data suggest the possibility that properties of mammalian thymidylate synthases may differ, and that kinetic characteristics of enzymes purified from different human sources may also vary. Cheng et al. (42) compared the inhibition of thymidylate synthases isolated from human AML cells or KB cells by N1’-propargyl-5,8-dideazafolate, and found that with CHz-HIPteGluI as the substrate, inhibition in the former was competitive, and in the latter, noncompetitive. K, values for [6R]CHz-HIPteGluI also differed (20 and 105 PM, respectively). The degree and type of inhibition therefore appeared to depend upon the source of the enzyme. Consequently, elucidation of how polyglutamates of CHa-H,PteGlu influence the interaction of FdUMP with thymidylate synthase isolated from the colon tumor is therefore an important area for subsequent investigation due to the importance of the enzyme as a target for 5fluorouracil in this disease.
4. 5. 6. 7. 8.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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