Fluorine-19 nuclear magnetic resonance investigation of the

The presence of the catalytic ternary complex has been inferred from studies of the inhibitory ternarycomplex in which 5-fluoro-2'-deoxyuridylate, FdU...
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Biochemistry 1980, 19, 116-123

Huang, C., & Carlton, J . (1971) J . Biol. Chem. 246, 2555-2560. Kao, Y. J., Charlton, S. C., & Smith, L. C. (1977) Fed. Proc., Fed. Am. SOC.Exp. Biol. 56, 936. Long, F. A., & McDevit, W. R. (1952) Chem. Rev. 51, 119 -169. Mabrey, S., & Sturtevant, J. M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3863-3866. Patel, K. M., & Sparrow, J. T. (1978) J . Chromatogr. 150, 542-547. Patel, K. M., Pownall, H . J., Morrisett, J. D., & Sparrow, J. T. (1976) Tetrahedron Lett. 45, 4015-4018. Pownall, H . J., & Smith, L. C. (1974) Biochemistry 13, 2594-2597 Rigaud, J., Cary-Bobo, C., Sanson, A., & Ptak, M. (1977) Chem. Phys. Lipids 18, 23-38. ~

Robinson, D. S. (1970) Compr. Biochem. 18, 51-116. Scow, R. O., Hamosh, M., Blanchette-Mackie, E. J., & Evans, A. J. (1972) Lipids 7, 497-505. Sengupta, P., Sackmann, E., Juhnle, W., & Scholz, J. P. (1976) Biochim. Biophys. Acta 436, 869-878. Sklar, L., Hudson, B. S., & Simoni, R. (1977) Biochemistry 16, 819-825. Smith, L. C., & Scow, R. D. (1979) Prog. Biochem. Pharmacol. 15, 65-91. Soutar, A. K., Pownall, H. J., Hu, A., & Smith, L. C. (1974) Biochemistry 13, 2828-2836. Vanderkooi, J., & Callis, J. (1974) Biochemistry 13, 4000-4006. Van Dijck, P., De Kruijff, B., Aarts, P., Verkleij, A,, & De Gier, J. (1978) Biochim. Biophys. Acta 506, 183-191.

Fluorine- 19 Nuclear Magnetic Resonance Investigation of the Noncovalent and Covalent Binary Complexes of 5-Fluorodeoxyuridylate and Lactobacillus casei Thymidylate Synthetase? Charles A. Lewis, Jr.,* Paul D. Ellis,$ and R. Bruce Dunlap*

ABSTRACT: Formation of the S-fluorodeoxyuridylate-thymidylate synthetase binary complex produced 19FN M R resonances 1.4 and 34.5 ppm to higher shielding of free nucleotide. Denaturation demonstrated that the greatly shifted resonance represented nucleotide covalently attached to the enzyme and afforded narrowing of the resonance so that it could be resolved into a doublet and a singlet. Comparison with 19F N M R spectra of bisulfite adducts of 5-fluorodeoxyuridine and 5fluorodeoxyuridylate permitted identification of the covalent species as the 5,6-dihydro derivative of 5-fluorodeoxyuridylate which is linked to the catalytic cysteine in the active site of the enzyme. The doublet and singlet resonances of the dihydro species result from the H 2 0 / D 2 0content of the buffer which produced an isotope-shifted 19Fresonance for the deuterated species. The enzyme appears to stereoselectively produce a

single isomer of the covalent binary complex. The extent of complex formation was observed to be dependent upon the concentration of phosphate present in solution, with high concentrations disfavoring complex formation. The greatest extent of binary complex formation was found in Tris-HC1 buffer. In piperazine-N,N’-bis(2-ethanesulfonic acid) (Pipes) buffer, significantly increased resolution of the resonance for the covalent species was obtained upon addition of less than an equivalent amount of phosphate to the binary complex. This permitted direct observation of the 5,6-dihydro species resonance in the native binary complex. Binding of 5fluorodeoxyuridylate to thymidylate synthetase occurs as an equilibrium mixture of two bound forms, a noncovalent Michaelis complex of the inhibitor with the enzyme and a covalent 5-fluoro-5,6-dihydrodeoxyuridylate-6-enzymecomplex.

S t u d i e s by numerous investigators permit us to write a moderately detailed chemical mechanism for the reductive methylation of dUMP’ catalyzed by thymidylate synthetase (EC 2.1.1.45) to form dTMP utilizing CHzHlfolate as the source of the methyl group (Scheme I) (Friedkin, 1973; Danenberg, 1977; Pogolotti & Santi, 1977). The central feature of this mechanism is the formation and breakdown of

a 5,6-dihydro derivative of dUMP which is covalently bound to both the enzyme and CH2H4folate [(C) of Scheme I]. The catalytic ternary complex results from attack of the active-site cysteine on carbon 6 of dUMP, followed by electrophilic addition at carbon 5 by the methylene group of CH,H4folate. Decomposition of this complex is initiated by abstraction of the proton on carbon 5, followed by either a concerted breaking of the remaining bond to the folate with internal hydride transfer (Friedkin, 1959) or formation of an exocyclic methylene group on carbon 5 , which then undergoes reduction (Santi et al., 1974).

From the Department of Chemistry, University of South Carolina, Columbia, South Carolina 29208. Received May 29, 1979. This work was supported in part by the National Institutes of Health through Grants CA 12842 and CA 15645 from the National Cancer Institute. * Correspondence should be addressed to this author. He is the recipient of a Faculty Research Award (FRA-144) from the American Cancer Society. *Recipient of a National Research Service award (CA 06097) from the National Cancer Institute of the National Institutes of Health. 5A fellow of the A. P. Sloan Foundation.

Abbreviations used: dUMP, deoxyuridylate; dTMP, deoxythymidylate; CH,H,folate, (f)-5,10-methylene-5,6,7,8-tetrahydrofolate; FdUMP, 5-fluorodeoxyuridylate; FdU, 5-fluorodeoxyuridine; MMTS, methyl methanethiolsulfonate; Pipes, piperazine-N,N’-bis(2-ethanesulfonic acid); 5-N02dUMP, 5-nitrodeoxyuridylate.

0006-2960180 10419-01 16$01.OOlO 0 1980 American Chemical Societv I

,

19F NMR OF FDUMP-DTMP

SYNTHETASE COMPLEXES

V O L . 1 9 , N O . 1, 1 9 8 0

117

to a form covalently bound to the enzyme. Comparison with bisulfite addition to FdU and FdUMP has allowed us to identify the covalent species as the 5,6-dihydro derivative of FdUMP linked to the enzyme through a sulfide bond at carbon 6. This species appears to result from the trapping of the carbanion intermediate (B) illustrated in Scheme I.

Scheme I

i

qq HW

The presence of the catalytic ternary complex has been inferred from studies of the inhibitory ternary complex in which 5-fluoro-2’-deoxyuridylate, FdUMP, has been substituted for dUMP. The interaction of enzyme, FdUMP, and CH2H4folatehas been shown to occur through covalent bonds in the intact enzyme (Langenbach et al., 1972; Danenberg et al., 1974) and in the isolated active-site peptide containing both these ligands (Sommer & Santi, 1974; Pogolotti et al., 1976; Danenberg & Heidelberger, 1975). Santi and co-workers (James et al., 1976; Santi et al., 1976) reported the first I9F N M R observations of FdUMP in the isolated active-site peptide and attempted to characterize the structure of the complex and how it was produced. Extensive 19FN M R studies performed in these laboratories (Byrd et al., 1977, 1978) on the native and denatured ternary complex in the intact enzyme resulted in elucidation of the relative stereochemistry of the folate and enzyme substituents on the 5-fluoro-5,6-dihydrouracil ring. The present study has further utilized I9F NMR to examine steps leading to the formation of the inhibitory ternary complex by examining the interaction of FdUMP with the enzyme in a binary complex. The formation of binary complexes of the enzyme with dUMP or FdUMP has been demonstrated by equilibrium dialysis (Galivan et al., 1976), circular dichroism (Leary et al., 1975; Plese & Dunlap, 1978), microcalorimetry (Beaudette et al., 1977), and Hummel-Dreyer equilibrium gel filtration (Beaudette et al., 1977; Plese et al., 1979), and we have reported our initial observation of the 19FNMR spectrum of FdUMP in a binary complex with the enzyme (Lewis et al., 1978a). The present study has shown that the 19FN M R spectrum of the thymidylate synthetase-FdUMP binary complex exhibits two resonances, one corresponding to noncovalent association of nucleotide and the other corresponding

Experimental Procedures Thymidylate synthetase was isolated in the presence of exogenous thiol from amethopterin-resistant Lactobacillus casei according to the procedure of Lyon et al. (1975). Enzyme emerging from the hydroxylapatite column at the end of the isolation is designated HA-thymidylate synthetase. The enzyme was also purified in the absence of phosphate by extensive dialysis in Tris-HC1 buffer, followed by DEAESephadex column chromatography as described by Byrd (1977). This phosphate-free preparation is referred to as DEAE-thymidylate synthetase. All enzymes employed in these studies were homogeneous by gel electrophoresis, yielded normal ternary complex gels (Aull et al., 1974; Donato et al., 1976), and exhibited specific activities in excess of 2.5 units/mg of protein (Wahba & Friedkin, 1961). Samples were prepared for N M R spectroscopy by concentrating a solution containing about 30 mg of purified enzyme in an Amicon ultrafiltration cell to a volume of about 4 mL for a final enzyme concentration of 80-100 pM. Activation was accomplished by dialysis in 2 L of 0.1 M buffer (Tris-HC1 or N a P 0 4 ) at pH 7.4 containing 1 mM EDTA and 25 mM 2-mercaptoethanol, with two final dialyses in 150 mL of the same buffer prepared with 30% deuterium oxide to provide a lock signal for the N M R spectrometer. Enzyme concentration was measured spectrophotometrically at 278 nm with the extinction of 1.05 X lo’ reported by Lyon et al. (1975). Binary complex samples were prepared by mixing a 2.5 molar excess of FdUMP with the enzyme and transferring the solution to an 18-mm flat-bottom N M R tube which was then fitted with a nylon vortex plug. Binary complexes were converted to ternary complexes by addition of a 10-fold excess of (f)CH,H,foIate as described by Byrd (1977). Samples were denatured by addition of a concentrated solution of sodium dodecyl sulfate to a final concentration of 1.2% (Byrd, 1977). Fluorine-19 N M R spectra were obtained at 94.1 M H z on the highly modified Varian XL-100-15 N M R spectrometer in the Department of Chemistry at the University of South Carolina by utilizing the 18-mm multinuclear probe (Byrd & Ellis, 1977). All data were obtained at 20 f 1 OC in the Fourier transform mode, using 60’ pulse widths, with an acquisition time of 0.4 s, acquiring 4K data points and transforming 8K data points. Spectral windows varied from 5000 to 10000 Hz as required for the particular experiment. All spectra were referenced to free FdUMP in the samples, which was observed at 88.9 ppm to higher shielding of 6 mM trifluoroacetic acid in deuterium oxide. Estimates of the percent composition of resonances in the spectra were obtained by cut and weigh measurements of the areas of the expanded resonances and were usually reproducible within 5-10% from sample to sample. Methyl methanethiolsulfonate, MMTS, was employed to prepare thymidylate synthetase in which the catalytic cysteine sulfhydryls were blocked by a CH3S- group (Lewis et al., 1978b). Concentrated DEAE-enzyme was activated by dialysis in 50 mM Pipes buffer, pH 7.4, containing 1 mM EDTA and 25 mM 2-mercaptoethanol and then dethiolated on a Sephadex G-10 column ( 2 X 24 cm). Pooled dethiolated enzyme yielded a specific activity of 2.0 units/mg by using

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ELLIS, AND DUNLAP

Table I: FdUMP Binding Stoichiometries in Binary Complexes with Thymidylate Synthetasea non-

enzyme and buffer DEAE-TS in Tris-HC1 DEAE-TS in Pipes HA-TS in Tris-HC1 HA-TS in phosphate

covalent FdUMP/ enzyme

covalent FdUMP/ enzyme

1.50

0.50

1.01 0.93

0.47

0.55

0.35 0.29

total bound FdUMP/ enzyme 2.0 1.48 1.28 0.84

a Spectra were recorded in 100 mM buffers at pH 7.4 as described under Experimental Procedures. Binding stoichiometries were obtained by the cut and weigh method of expansions of resonances, calculation of their percentage of the total fluorine content of the spectrum, and calculation of the corresponding concentration of FdUMP. Division by the enzyme concentration yielded the ratio of FdUMPienzvine in the table above.

0

-I

PPM 1: High-resolution fluorine-19 NMR spectrum of 6 mM FdUMP in 0.1 M Tris-HC1, pH 7.4, 1 mM EDTA, and 25 mM 2-mercaptoethanolbuffer containing 90% deuterium oxide. The large doublet of doublets is 5’-FdUMP, with the doublet at -0.3 ppm being its 6-deuterio species. The other smaller resonances correspond to either 3’-FdUMP or FdU and its corresponding 6-deuterio species. This sample is 90% 5’-FdUMP. FIGURE

thiol-free CH2H4folate. A 14-fold excess of an aqueous MMTS solution was mixed with the enzyme at 0 “C, and assays were performed until inactivation was complete at 3 min. Unreacted MMTS was removed by passage through a second Sephadex G-10 column (3.5 X 23 cm). Fractions containing CH3S-thymidylate synthetase were concentrated to a volume of 5 mL and dialyzed against thiol-free Pipes buffer containing 30% DzO. The binary complex sample of this blocked enzyme was prepared in the usual manner and was examined by I9FNMR. For comparison, a native binary complex was prepared in Pipes buffer to determine the normal extent of complex formation under these conditions. The FdUMP employed in these studies was prepared by chemical phosphorylation of FdU (Sigma Chemical Co.) and chromatographed on Dowex 1-X10, 200-400 mesh, to purify the 5’ isomer (Dawson et aI., 1977). Samples of FdUMP were shown to be at least 90% the 5’-phosphate isomer by I9Fand )‘P N M R analysis. A typical high-resolution I9F N M R spectrum of FdUMP is shown in Figure 1. The 5’-FdUMP is resolved into a doublet of doublets (JHaF = 6.3 Hz; J H , , F = 1.7 Hz). At -0.3 ppm a small doublet is observed (JH,,F = 1.7 Hz), which is due to the 6-deuterio form of 5’-FdUMP. In this particular sample, smaller resonances appear at -0.9 and -1.2 ppm with the same coupling constants as described above and correspond to the 6-protio and 6-deuterio species of 3’-FdUMP or FdU. The deuteration results from dissolving the chelexed and lyophilized FdUMP samples in D 2 0 for 31P and I9F analysis and storing these frozen solutions until needed for binary complex formation. These slightly basic conditions appear sufficient to catalyze the deuteration of FdUMP at carbon 6 (Cushley et al., 1968). The presence of 3’-FdUMP and FdU is due to decomposition of the 5’ isomer through isomerization and dephosphorylation, respectively. The small resonance observed in some of the spectra in this paper to slightly higher shielding of FdUMP is therefore either 6deuterio-FdUMP or 3’-FdUMP and is most visible when the free ligand resonance is sharpened by the addition of NaDodS04 (Figure 3). Any contribution that this species might

have to the resonances observed for the 5’ isomer in the noncovalent and covalent complexes is negligible. Bisulfite adducts of FdU and FdUMP were prepared by dissolving the nucleoside or nucleotide in neutral solutions of sodium bisulfite in H,O, D 2 0 , or H 2 0 / D 2 0mixtures as described for 5-fluorouracil (Sedor et al., 1974). The reaction mixtures were lyophilized and stored as the dry solid until redissolved in either D 2 0 or H 2 0 containing 10% D 2 0 for a lock signal. The I9F spectra of the adducts were referenced to the chemical shift of FdUMP for direct comparison with the binary complex spectra. Results The I9F N M R spectrum of FdUMP in the presence of thymidylate synthetase exhibited resonances at -1.4 and -34.5 ppm from free FdUMP as shown in Figure 2. The slightly shifted resonance, whose line width is somewhat greater than that of free ligand (36 Hz compared to 24 Hz), represents noncovalently bound nucleotide as previously reported (Lewis et al., 1978a). However, the approximately 100-Hz-wide resonance which exhibited the large chemical shift change from free FdUMP was found to be covalently attached to the enzyme, since the resonance remained after addition of sodium dodecyl sulfate (Figure 3). Denaturation also produced a significant narrowing of the resonance of the covalent species, which allowed it to be resolved into a doublet ( J = 45-50 Hz) and a singlet. Figure 2 also illustrates the effect of phosphate on the extent of binary complex formation. The quantitation obtained from these spectra for the amounts of FdUMP as noncovalent complex and covalent complex is shown in Table I. DEAEthymidylate synthetase, which has been shown to be free of bound phosphate by 31PNMR,2 exhibited the greatest extent of complex formation with FdUMP in Tris-HC1 buffer (Figure 2A). HA-enzyme in phosphate buffer bound the least amount of nucleotide, under otherwise comparable conditions (Figure 2D). Interestingly, removal of excess phosphate from the HA-enzyme by extensive dialysis in Tris-HC1 resulted in binding of an intermediate amount of FdUMP when the complex was examined in Tris-HC1 buffer (Figure 2C). This situation was also observed when the binary complex was examined in Pipes buffer (Figure 2B). Variation of the phosphate buffer concentration from 100 to 5 mM produced an increase in binary complex formation as shown in Figure 4.

Addition of CH2H4folate to the native binary complex formed in Tris-HC1 buffer resulted in the appearance of a R. A. Byrd, P. D. Ellis, and R. B. Dunlap, unpublished results.

I9F NMR OF FDUMP-DTMP

VOL.

SYNTHETASE COMPLEXES

A

19, NO.

1, 1980

119

B

PPM

PPM

2: Fluorine-19 N M R spectra of thymidylate synthetase-FdUMP binary complexes. (A) DEAE-enzyme in 0.1 M Tris-HC1. (B) DEAE-enzyme in 0.1 M Pipes. (C) HA-enzyme in 0.1 M Tris-HC1. (D) HA-enzyme in 0.1 M phosphate. All spectra were recorded at p H 7.4 and 20 O C and are the result of 200000 or more transients. Chemical shifts are referenced to free FdUMP at 0 ppm. FIGURE

II

J J 0

-lo

-20

-30

- 40

PPM

FIGURE 3: Fluorine-19 N M R spectrum of the thymidylate synthetase-FdUMP binary complex denatured by addition of sodium dcdecyl sulfate to a final concentration of 1.2% in 0.1 Pipes, p H 7.4. Note the increased resolution of the covalent complex resonance at about -36 ppm. The inset is a twofold increase in the horizontal and vertical scale of the covalent resonance, showing the coupling of about 45 Hz.

broad resonance (line width 96 Hz) in the I9FNMR spectrum at -13 ppm which is characteristic of the ternary complex of

enzyme-FdUMP-CHzH4folate (Figure 5 ) (Byrd, 1977; Byrd et al., 1978). The ternary complex spectrum was observed with concurrent loss of the two resonances corresponding to the binary complex, indicating that the binary complex is not a dead-end or abortive complex of the enzyme. Denaturation of the native ternary complex produced the expected shift of the ternary complex resonance to -1.7 ppm (Byrd, 1977; Byrd et al., 1978). MMTS-inactivated thymidylate synthetase, in which the catalytic cysteine was blocked in a mixed disulfide with CH3S-, was found to be incapable of forming a binary complex with FdUMP as judged by I9F NMR. Spectra of the attempted complex were accumulated to comparable levels of signal to noise as normal binary complexes, yet no resonance for the noncovalent or the covalent complex was observed. Similar results have been obtained by Munroe and D ~ n l a p , ~ who attempted the examination of FdUMP binding to MMTS-inactivated thymidylate synthetase by circular dichroism as performed by Plese & Dunlap (1978). These observations suggest that the catalytic integrity of the cysteinyl thiol is required for the enzymic active site to be competent. The 19F N M R spectra of the FdU and FdUMP bisulfite adducts are presented in Figure 6. In pure HzO, the bisulfite adduct fluorine resonances were found to be a pair of doublets W. A. Munroe and R. B. Dunlap, unpublished results.

19F NMR O F FDUMP-DTMP

VOL. 19, NO. 1 , 1980

SYNTHETASE COMPLEXES

nonequivalent isomers of the bisulfite adduct of FdU due to the presence of four stereoisomers of the adduct. This nonequivalence is responsible for the resonance doubling observed in the 19FN M R spectra of the adducts shown in Figure 6. Discussion The 19FN M R spectrum of the 5-fluorodeoxyuridylatethymidylate synthetase binary complex is composed of both covalent and noncovalent species. This result provides some explanation for the extremely tight binding of nucleotides to the enzyme. The small changes in line width and chemical shift of the resonance for the noncovalently bound nucleotide suggest that it has undergone minor alterations from free FdUMP in solution. Although such small fluorine chemical shift changes are often difficult to interpret, there are two possibilities that are consistent with the observed shift. Byrd et al. (1977) reported that when the ternary complex is prepared by using 5,2’-difluoro-2’-deoxyuridylate(F,dUMP), the 19F resonance of the 2‘-fluorine shifted 2.4 ppm to higher shielding from the free ligand, reflecting changes in the microenvironment of the fluorine nucleus. Alternatively, Leary et al. (1975) have interpreted circular dichroism changes associated with d U M P binding to thymidylate synthetase to be due to rotation of the pyrimidine ring about the glycosidic bond from the anti conformation toward the syn conformation. Such a rotation might be expected to position carbon 6 for attack by the enzymic catalytic cysteine. Plese & Dunlap (1978) have observed similar circular dichroic spectral changes upon FdUMP binding to the native enzyme. If such a rotation occurred in forming the noncovalent portion of the binary complex, the fluorine nucleus would be moved away from the 5’-phosphate group with a slight increase in chemical shift to higher shielding. Despite the specific details of the interaction, the noncovalent complex appears to represent a Michaelis type complex of FdUMP with the enzyme. In contrast, the covalent portion of the binary complex has undergone considerable change from free FdUMP as indicated by the greatly increased line width of the resonance and large change in chemical shift to higher shielding. The presence of a covalent species in the thymidylate synthetase-FdUMP binary complex 19FN M R spectrum suggested that the enzymic catalytic cysteine had attacked the 6 position of the pyrimidine ring. Further, the observation of a coupling constant of about 45 H z in Figure 3 was consistent with both a proton and fluorine on carbon 5, as reported for 5-fluoro5,6-dihydrouracil-6-sulfonate(Sander & Deyrup, 1972). In order to test the hypothesis that a 5-fluoro-5,6-dihydrouridylate species might be covalently bound to the enzyme, we examined the I9FNMR spectra of bisulfite adducts of FdU and FdUMP to determine if this model could account for chemical shift and doublet and singlet resonances observed for the denatured binary complex. The production of two enantiomeric forms of the bisulfite adduct of FdU resulted in the doubling of the fluorine resonances in Figure 6 . These spectra revealed that the formation of the 5,6-dihydro derivative of FdU by bisulfite addition produced resonances with chemical shifts within 5 ppm of those observed for the denatured binary complex and comparable multiplicity (compare the inset of Figure 3 with the X and X‘ resonances in Figure 6B). As one might have predicted, the enzyme generated only one of the two magnetically nonequivalent isomers through stereoselective addition to the nucleotide. Deuteration of carbon 5 of the bisulfite adducts produced the singlets observed to higher shielding of each of the protonated adduct resonances. In the native binary complex, the covalent resonance is asymmetric in shape, suggesting a complex structure which is hidden by loss of

121

A

-io

-20 PPM

-30

L -40

B

PPM

FIGURE7: Fluorine- 19 NMR spectrum of the binary complex in 0.1

M Tris-HCI, pH 7.4, buffer containing 90% D 2 0 native (A) and denatured by addition of NaDodS04 to 1.2% (B). Note the exclusive production of the 5-deuterated form of the 5,6-dihydro-S-fluorodeoxyuridylate covalently linked to the enzyme and its similarity to the spectrum in Figure 6C. The small resonances in (B) near the free FdUMP resonance are the contaminants illustrated in Figure 1.

resolution due to slow exchange with noncovalently bound and free FdUMP and the slow correlation time of the enzyme. Denaturation of the complex stopped the exchange process, unfolded the tertiary structure of the enzyme, and revealed the underlying structure of the resonance to be a doublet and singlet in a ratio of about 2:1, of the protio and deuterio isomers of the dihydro species, which reflected the 30% D 2 0 content of the buffer. Observation of the binary complex in Tris-HC1 buffer prepared with 90% D 2 0 produced a covalent resonance that was greatly reduced in line width (Figure 7A). Upon denaturation, only a singlet was observed for the covalent complex resonance a t a chemical shift identical with that observed for the singlet in the denatured binary complex in buffers containing only 30% D 2 0 (compare Figure 7B to Figure 3 inset). Therefore, the covalent species of the binary complex appears to be a 5-fluoro-5,6-dihydrouridylatespecies which is covalently linked to the enzyme through a sulfide bond at carbon 6 and carries a proton or deuteron on carbon 5. The 5,6-dihydro species apparently was produced from the reactive carbanion intermediate (Scheme IB) which would normally

122

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LEWIS, ELLIS, AND DUNLAP

Scheme I1

ow

0

undergo reaction with the methylene carbon of CH2H4folate to form the ternary complex. Sedor et al. (1974) have elucidated the mechanism of bisulfite addition to 5-fluorouracil as shown in Scheme 11. The enolate produced by bisulfite attack on carbon 6 (Scheme IIB) is a resonance form of the carbanion (Scheme IB) in the enzyme reaction. Enzymic attack on carbon 6 would generate the carbanion, which, in the absence of CH2H4folate,could shift electrons to generate the enolate which would lead to formation of the 5,6-dihydro derivative of FdUMP through a series of reactions analogous to those in Scheme 11. Although the 5-fluoro-5,6-dihydrodeoxyuridylate-6-enzyme complex survived denaturation, it was observed to undergo a slow hydrolysis which liberated FdUMP and denatured enzyme. Presumably, this reaction was initiated by proton abstraction from carbon 5 , in a reversal of Scheme I1 as proposed for decomposition of bisulfite adducts of 5-fluorouracil (Sedor et al., 1974). Observation of two 19FN M R resonances for the FdUMPthymidylate synthetase binary complex might be argued to indicate the presence of two binding sites for FdUMP, with different affinities, as proposed by others on the basis of binding studies (Galivan et al., 1976). Estimated binding stoichiometries from the I9F N M R spectra, shown in Table I, demonstrate that under the most favorable conditions for complex formation (in Tris-HCI and Pipes buffers) an average of 1.6 FdUMP molecules is bound per enzyme dimer. This value is comparable to the 1.7 FdUMP binding sites determined by ternary complex formation (Aull et al., 1974; Donato et al., 1976), binary complex circular dichroism studies (Plese & Dunlap, 1978), and catalytic sulfhydryl group modification (Plese & Dunlap, 1977; Lewis et al., 1978b) of thymidylate synthetase isolated in the presence of exogenous thiol. In the circular dichroism study mentioned above, no low-affinity sites were found as nucleotides were titrated into the enzyme. The I9FN M R data suggest that the FdUMP-enzyme binary complex is an equilibrium mixture of two forms of the nucleotide bound at equivalent sites, differing in only the presence or absence of a covalent bond. The reversibility of the binary complex was demonstrated by the ability to form the inhibitory ternary complex by addition of CH2H4folate. The I9F N M R spectrum obtained was characteristic of the ternary complex and contained no binary complex resonances, noncovalent or covalent. Therefore, the covalent species of the binary complex does not represent a dead-end form of the enzyme but rather a form which is chemically related and

readily converted to the carbanion intermediate required for ternary complex formation. The inability to generate a binary complex spectrum with enzyme in which the catalytic cysteine was blocked by a CH3S- group indicated that FdUMP would not bind to the enzyme nonspecifically as might be expected for a low affinity site. Further, this experiment indicated that when the catalytic cysteines were chemically blocked, FdUMP could not bind either noncovalently or covalently to the enzyme, as viewed by I9FNMR. Studies are continuing in our laboratory to demonstrate the equivalent binding of nucleotides in binary complexes. In phosphate buffer, the percentage of FdUMP bound noncovalently and covalently to the enzyme is greatly reduced (see Table I). These results apparently reflect increased dissociation constants for FdUMP from the binary complex as measured by equilibrium dialysis in phosphate as compared to Tris-HC1 (Galivan et al., 1976). Phosphate appears to inhibit formation of the FdUMP-thymidylate synthetase binary complex. A priori, it might be suggested that this reflected competition for the 5’-phosphate binding site within the active site; however, phosphate does not protect arginine residues which are involved in nucleotide binding from chemical m~dification.~Phosphate has been shown to produce pronounced reductions in the rate of catalytic cysteine modification (Lewis et al., 1978b). Phosphate may interfere with formation of the covalent portion of the binary complex, through some modulation of the reactivity of the active-site cysteine, which may lead to an overall decrease in FdUMP binding. Phosphate was shown to be ineffective in disrupting a preformed binary complex in concentrations up to a 10-fold excess over the enzyme. In these studies, addition of an equimolar concentration of phosphate reproducibly produced a distinct narrowing of the covalent resonance of the native binary complex, which partially resolved the doublet and singlet structure described above for the denatured binary complex (compare parts A, B, and C of Figure 8). This partial increase in resolution of the covalent resonance occurred without a change in chemical shift, which is indicative of a possible slowing of the exchange rate of nucleotide out of the covalent complex. This observation confirmed the assignment of the 5,6-dihydroderivative of FdUMP as the covalent species of the binary complex. At this time we are unable to more clearly delineate the source of the effects produced by phosphate, but 31Pexperiments to be performed at 81 and 162 MHz will enable us to examine the interaction of nucleotides and phosphate with the enzyme in more detail. Fluorine-19 NMR has enabled us to determine that FdUMP is bound both noncovalently and covalently in the binary complex with thymidylate synthetase. The noncovalent species appears to be a Michaelis complex of the inhibitor on the enzyme in which a partial rotation of the pyrimidine ring about the glycosidic bond may have occurred. The covalent species, which is bound to the enzyme through a sulfide bond at carbon 6, has been identified as the 5,6-dihydro derivative of FdUMP, which results from trapping of the carbanion intermediate (Scheme IB) which normally would react with CH2H4folate to produce the ternary complex. Recently Matsuda et al. (1978) have reported that 5NOzdUMP is a potent inhibitor of thymidylate synthetase, in the absence of CH,H,folate. These workers attributed the inhibition to the formation of a binary complex of 5NOzdUMP and the enzyme, with the nitro group serving to stabilize the carbanion intermediate. The data presented in ~~~~~

K . L. Cipollo and R. B. Dunlap, unpublished results

19F N M R O F FDUMP-DTMP

SYNTHETASE COMPLEXES

A

C

-30

-35

-40

PPM FROM FDUMP FIGURE 8: Fluorine-19 N M R spectrum of the covalent portion of the binary complex in 0.1 M Pipes buffer, p H 7.4 (A), after addition of 1 equiv of phosphate (B), and after denaturation by addition of NaDodSOl to 1.2% (C). The partial increase in resolution in (B) reveals some of the structure of the covalent resonance otherwise observed only under denaturing conditions (C).

this paper would suggest that this complex may in fact be the 5,6-dihydro derivative of 5-N02dUMPbound to the active-site cysteine, as described for the FdUMP-thymidylate synthetase binary complex. References Aull, J. L., Lyon J. A., & Dunlap, R. B. (1974) Microchem. J. 19, 210-218. Beaudette, N. V., Langerman, N., Kisliuk, R. L., & Gaumont, Y. (1977) Arch. Biochem. Biophys. 179, 272-278. Byrd, R. A. (1977) Ph.D. Thesis, University of South Carolina. Byrd, R. A., & Ellis P. D. (1977) J . Magn. Reson. 26, 169-173. Byrd, R. A,, Dawson, W. H., Ellis, P. D., & Dunlap, R. B. (1977) J . Am. Chem. Soc. 99, 6139-6141. Byrd, R. A,, Dawson, W. H., Ellis, P. D., & Dunlap, R. B. (1978) J . Am. Chem. Soc. 100, 7478-7486. Cushley, R. J., Lipsky, S . R., & Fox, J. J. (1968) Tetrahedron Lett., 5393-5396. Danenberg, P. V. (1 977) Biochim. Biophys. Acta 473, 73-92.

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