Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2′-deoxyuridylate and 5,10-methylenetetrahydrofolate

J. Mol. Biol. (1990)

214,

937-948

Stereochemical Mechanism of Action for Thymidylate Synthase Based on the X-ray Structure of the Covalent Inhibitory Ternary Complex with 5-Fluoro-2’-deoxyuridylate and 5,10-Methylenetetrahydrofolate D. A. Matthews,

J. E. Villafranca, C. A. Janson, W. W. Smith K. Welsh and S. Freer Agouron Pharmaceuticals, Inc. 11025 N. Torrey Pines Road La Jolla, CA 92037, U.S.A.

(Received 20 November 1989; accepted 14 April

1990)

The structure of the Escherichia coli thymidylate synthase (TS) covalent inhibitory ternary complex consisting of enzyme, 5-fluoro-2’-deoxyuridylate (FdUMP) and 5,10-methylene tetrahydrofolate (CH,-H,PteGlu) has been determined at 2.5 A resolution using difference Fourier methods. This complex is believed to be a stable structural analog of a true catalytic intermediate. Knowledge of its three-dimensional structure and that for the apo enzyme, also reported here, suggests for the first time how TS may activate dUMP and CH,-H,PteGlu leading to formation of the intermediate and offers additional support for the hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the substrate nucleotide and N-5 of the cofactor. By correlating these structural results with the known stereospecificity of the TS-catalyzed reaction it can be inferred that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the bond linking C-5 of dUMP to C-11 of the cofactor. The elimination itself may be catalyzed by proton transfer to the cofactor’s 5 nitrogen from invariant Asp169 buried deep in the TS active site. The juxtaposition of Asp169 and bound tetrahydrofolate in TS is remarkably reminiscent of binding geometry found in dihydrofolate reductase where a similarly conserved carboxyl group serves as a general acid for protonating the corresponding pyrazine ring nitrogen of dihydrofolate.

1. Introduction

regard has focused on one particularly close structural analog of the catalytic ternary complex, namely the complex between TS, CH,-H,PteGlu and 5-fluoro-2’.deoxyuridylate (FdUMP), a metabolite of the antipyrimidines 5-fluorouracil and 5-fluorodeoxyuridine. It has been suggested that in the presence of CH,-H,PteGlu, FdUMP acts as a mimic of the substrate and is committed to the catalytic reaction up to the point where it forms a stable covalent adduct with an active site nucleophile but proceeds no further because F+, unlike a proton, cannot be extracted by the enzyme to force collapse of the enzyme-FdUMP-CH,-H,PteGlu ternary complex leading to products (Santi & Danenberg, 1984, and references cited therein). In certain tumors the activity of fluorouracil is enhanced by concurrent administration of 5-formyl-H,PteGlu, which is converted metaboli-

Friedkin (1959) proposed that a key intermediate in the thymidylate synthase (TSt)-catalyzed reacmethylene bridge tion contains a covalent between C-5 of 2’-deoxyuridylate (dUMP) and N-5 of 5: lo-methylenetetrahydrofolate (CH,-H,PteGlu; Fig. 1). Studies of inhibitory ternary complexes that mimic the proposed intermediate have been important in providing a basis for our current understanding of the enzyme’s mechanism of action (Cisneros et al., 1988). Much recent effort in this

tAbbreviations used: TX; thymidylate dUMP, 2’-deoxyuridylate; CH,-H,PteGlu; 5,10-methylenetetrahydrofolate. FdUMP, 2’-deoxyuridylate; PDDF, lo-propargyl5;8-dideazafolate; DHFR, dihydrofolate

0022%2836/90/160937-12 $03.00/O

synthase; Sfluoro-

reductase.

937 0

1990 Academic

Press Limited

D. A. Matthews

938 Pteridine

ring

L-Glutamate

p-Aminobenzoyl

et al.

structures permit identification of protein conformational changes that may be important for eatalysis and provide new insights into the enzymatic mechanism of action.

2. Structure Determination of Apo E. COB Thymidylate Synthase 5,10-Methylenetettahydrofolate

IO-Propargyl-5,8-dideazafolate

otl c--P=0

b 5-Fluoro-2’-deoxyuridylate

Figure 1. Covalent structure for 5,10-methylenetetrahydrofolate 5&dideazafolate and FdUMP.

and atom numbering (top), lo-propargyl-

tally to CH,-H,PteGlu, the cofactor for TS catalysis. This observation suggests t,hat for these tumors, of the TX-FdUMP-CH,-H,PteGlu formation ternary complex is responsible for the cytotoxic effects of fluoropyrimidines (Berger & Hakala, 1984; Houghton et al., 1986). Thus, in addition to its possible mechanistic significance as a stable analog of a transient’ enzyme steady-state intermediate, the covalent inhibitory ternary complex is of interest because an understanding of how the folate cofactor modulates the interaction of FdUMP with TS could stimulate novel ways of thinking about how to inhibit the enzyme. In the previous paper (Matthews et aZ., 1990) we discussed the binding of FdUMP and lo-propargyl-5,8-dideazafolate (PDDF) to Escherichia coli TS. In what follows we present X-ra.y structures for the apo enzyme and for the covalent inhibitory ternary complex. Comparisons between these two

TS was isolated from E. ~oli transformed with a high amplifying expression plasmid containing the E. coli thymidylate synthase gene. The plasmid was constructed by subcloning the E. coli TS gene from plasmid pKTAH (Belfort et aE., 1983) via a BcZI restriction site created by site-directed mut,agenesis into a plasmid conta.ining t,be E. coli fol promoter. Crystals of E. coli TS apo-protein were grown from a solution of 34 mg of protein/ml at room temperature using the hanging drop method of vapor diffusion. The precipitant solution contained 60 miv-Tris (pH 8.5). 1% w-ammonium sulfate. Crystals appeared in less than 1 week and were dodecahedral in morphology with dimensions routinely as large as 0% mm on an edge. Data from a single native crystal were collected on a Rigaku AFC-6 diffractometer equipped with a graphite monochromator and a dual chamber area det,ector system of the Xuong-Hamlin design (San Diego Multiwire Systems). A data set consisting of 75,393 observations of 13,687 unique reflections to a resolut’ion of 2.5 A (1 A =O.l nm) was measured from 1 crystal in approximately 36 h. The crystals diffract strongly to at least 2-O a and the data are consistent with a bodycentered lattice in the cubic Laue group m3 w&h a= 133.0 8. The scaling R-factor on intensities for this data was 0.062. A second crystal was soaked in a small amount of HgUTP for 4 days. The data from this cryst,al contained 50,106 measurements of 12,995 unique refections between 25 and 2.5 8. The scaling R-factor on intensities for this data set was 0041. The native and KgUTP data were scaled together using Wilson scaling and the difference R-factor on intensities to 2.5 A was @16. A Patterson search at 2.5 a was carried out using an automated search procedure (Terwilliger et al., 1985) in space groups 123 and 12,3. No consistent solution was found in space group 123, in space group 12,3 two sites were located. This assignment of space group results in a V, value of 35 (approx. 68% solvent) for 1 subnunit of molecular weight 28,000 in the asymmetric unit and requires tha,t a crystallographic dyad axis relate the 2 subunits in the dimeric enzyme. Refinement of the heavy-atom parameters using the method of correlation of origin-removed Patterson functions (Terwilliger & Eisenberg, 1987) including the anomalous scattering for the Hg atom gave the foilowing parameters: Occupancy 0.37 0.17

x 0331 0.347

Y 0.186 @170

z

B

0.227 0,258

244 I%7

Mean figure of merit=O-36 Centric R = 054.

This solution was used to initiate 3 rounds of solventflattening utilizing the approach of Wang (1985). The procedure wa,s run twice, once with the above solution (enantiomer 1) and once with the enantiomorphic heavyatom constellation (enantiomer 2). The results of the 2 series of solvent-flattening cycles are given below.

Mechanism

Figure of merit R-factor Phase shift (deg.) Correlation

Enantiomer 078 0.22 4300 0.97

1

of Action for Thymidylate

Enantiomer 2 0.76 0.30 44.00 0.94

The phases resulting from the combination of phases calculated from the Wang procedure (enantiomer 1) and the original SIRAS phases were used to calculate an electron density map at 2.5 ,& resolution. This map was readily interpreted. The HgUTP binding sites were located near Cys192 at the surface of the monomer. The separation of the 2 mercury atoms is only 5.1 8, indicating that the binding probably occurs at 2 different orientations of the Cys sulfur atom. No significant density for HgUTP was observed at the active site of the enzyme. Co-ordinates for the TS apo enzyme structure were obtained as follows. Two helices and 1 P-strand from our refined model of the TS-FdUMP-PDDF ternary complex (Matthews et al., 1990) were moved as separate atomic groupings into the apo enzyme unit cell and fit to their respective electron density using rigid-body translations and rotations. A least-squares matching of these co-ordinates to those for corresponding atoms in the original ternary complex with FdUMP and PDDF provided an initial mapping for transforming the complete set of ternary TS co-ordinates into the new cubic unit cell. This preliminary model was subsequently adjusted on a molecular graphics terminal in order to optimize the fit of all atoms to the experimental apo enzyme electron density. The resulting model had a erystallographic R-factor of @35 and was used to initiate structural refinement using the simulated annealing methods of Brunger (1988). Our current model contains all main-chain and side-chain atoms for a complete 264 amino acid residue subunit of E. coli TS and has a crystallographic R-factor of @22 for all diffraction data between 25 and 2.5d.

3. Structure of the Covalent Inhibitory Ternary Complex A binary complex of E. coli TS with FdUMP was prepared as described (Matthews et al., 1990) and incubated with a 35fold molar excess of H,PteGlu and formaldehyde. The resulting covalent inhibitory ternary complex was then crystallized using a hanging drop method and conditions similar to those reported by us for other E. coli TS ternary complexes (Matthews et al., 1990). X-ray diffraction data were collected as described for the apo enzyme yielding a total of 101,050 measurements of 30,997 unique reflections representing 91 o/o of all possible data to a resolution of 2.5 8. The scaling R-factor on intensities for this data was 0.072. The covalent inhibitory ternary complex is crystallographically isomorphous with the TX-FdUMP-PDDF structure reported in the accompanying paper (Matthews et al., 1990). The binding geometries for FdUMP and CH,-H,PteGlu were arrived at by interpreting difference maps calculated using coefficients (FaT - FpDDF)eiaPDDF and (21”,,,-FF,DDF)ei”PDDF (Fig. 2). In this notation F CIT and refer to observed structure factor FPDDF amplitudes for ternary complexes with CH,-H,PteGlu and PDDF, respectively, and

Synthase

939

crPDDF is a calculated phase for the TS-FdUMP-PDDF complex based on our current refined model for that structure. As expected, FdUMP and CH,-H,PteGlu bind at the TS active site in an orientation similar to that observed for the corresponding inhibitors in the ternary complex with PDDF (Fig. 3). There are, however, important differences particularly in the way the folate cofactor interacts with FdUMP. Since we have reported on the binding of FdUMP and PDDF to TS, our description of the TS-FdUMP-CH,-H,PteGlu ternary complex will focus on a discussion of differences between these two structures. The most significant peak in the difference map (unless otherwise noted, difference map refers to a map calculated with coefficients (F,-I-FPDDF)eibPDDF ) is a positive envelope of density 14 standard deviations (cr) above background connecting N-5 of CH,-H,PteGlu with C-5 of FdUMP. We assign this feature to a methylene bridge linking the folate’s pteridine ring to the pyrimidine ring of bound nucleotide. Covalent tethering of these two ligands at the TS active site pulls the folate cofactor’s pyrazine ring closer to the pyrimidine of FdUMP and reduces its thermal mobility compared to the corresponding part of PDDF. This interpretation is further supported by extensive positive difference density (11~) surrounding the benzene portion of PDDF extending down into the region between the quinazoline and the up-pointing face of the nucleotide’s pyrimidine. Weaker corresponding negative features are situated adjacent to the benzene’s distal face. The fully reduced pyrazine ring of CH,-H,PteGlu bound to TS is puckered in a half chair conformation with the C-6-C-9 bond axial and the C-6 hydrogen equatorial (Figs 2 and 3). It follows that the C-9-N-10 bridge between the heterocycle and the benzoylglutamate side-chain is oriented differently than in PDDF where the C-G-C-9 bond lies in the plane of the aromatic quinazoline ring. Thus, the cofactor’s C-9 and N-10 atoms are positioned above the pyrazine ring rather than approximately coplanar with it and off to one side. Atom coordinates for C-9 and N-10 in the two structures differ by about 2 d. One consequence of these structural differences is that the benzoylglutamate portion of the cofactor is on average about 1A closer to Va1262 near the protein’s carboxyl terminus than is the corresponding moiety in PDDF when bound to TS. Small (< 1 8) compensating changes in protein structure occur at Glu58, Ile79 and along the edge of helix VII where it abuts the active site, involving particularly residues 171, 172, 176 and 180. The side-chain of Glu58 apparently tracks the differences in position for N-10 in the two complexes as evidenced by corresponding positive and negative difference density symmetrically disposed on opposite sides of the Glu58 carboxylate group as positioned in the PDDF ternary complex. This change in position for Glu58 ,is associated with the appear-

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A. Matthews

et

ai.

Figure 2. Electron density for the covalent inhibitory ternary complex at 25 A resolution eaicuiated using coefficients (2F,,, - FPDDF)eiDPDDF,where F,,, and Frnnr refer to observed structure factor amplitudes for ternary complexes with $10.methylenetetrahydrofolate and lo-propargyl-5,8-dideazafolate, respectively, and crPDDF is a calculated phase for the TX-FdUMP-PDDF complex based on our current refined model. ante of difference density (60) for a new fixed solvent molecule hydrogen-bonded both to Glu58 and WaMOl and proximate (approx. 3.5 A) to N-10 of CH,-H,PteGlu and O-4 of FdUMP. A distinguishing feature of the PDDF inhibitor is the N-10 propargyl substituent. As expected there is substantial negative density in the difference map associated with change of the propargyl group in PDDF to a hydrogen in CH,-H,PteGlu. Adjacent to one edge of this negative trough and close to the folate cofactor’s O-4 is a broad slightly elongated peak (100) that represents difference density associated with a new location for the 4 0x0 group owing to a slight rotation of the pteridine ring about an axis perpendicular to the plane of its pyrimidine ring. Also contributing to the buildup of density at this location is the aforementioned repositioning of the C-9-N-10 bridge. Turning now to an examination of difference density in the vicinity of FdUMP, there are two important features to discuss. A 60 peak of positive electron density lies between the side-chain sulfur of Cysl46 and C-6 of the nucleotide. The simplest interpretation of this observation is that an equilibrium

Figure 3. Thymidylate methylenetetrahydrofolate shown in red.

synthase covalent (blue). Covalent bonds

exists between covalent and non-covalent forms of FdUMP bound to T’S, and that the proportion of nucleotide covalently linked to the active site cysteine residue can va,ry depending on the identit,y of the bound ligands. If bound CH,-H,PteGlu is more effective than PDDF in shifting the equilibrium between these two forms and increasing the total amount of FdUMP covalently linked to the enzyme, positive difference density would appear in the binding region between C-6 of FdUMP and the sulfur of Cys146 in accord with the experimental result. This interpretation is in good agreement with results from “F nuclear magnetic resonance and trichloroacetic acid trapping experiments for FdUMP bound to Lactobacillus casei TS where it was shown that folates modulate the dynamic partitioning between covalent and non-covalent forms and that covalent FdUMP binding is increasingly enhanced as the pyrazine ring of the folate molecule becomes more reduced (Lewis et al., 1981; Moore ef al., 1986). Another difference in the way FdUMP binds in these two ternary complexes is suggested by an elongated lobe of positive difference density (60) in

inhibitory ternary complex linking FdUMP to the cofactor

with FdUMP and to conserved

(green) Cys146

and (yellow)

5>10are

Mechanism

of Action for Thymidylate

the vicinity of N-3, C-4 and O-4 slightly below the nucleotide’s pyrimidine ring as bound in the TS-FdUMP-PDDF complex. This feature, in conjunction with the presence of a weakly negative region nearby, indicates that O-4 and to a lesser extent N-3 and C-4 rotate away from the folate’s pyrazine ring upon covalent bond formation between the bridging methylene and C-5 of bound FdUMP. We attribute this change in geometry to accentuated puckering of the fluorouracil ring that occurs when the C-5-C-6 bond in a conjugated pyrimidine is fully saturated (Suck et al., 1972). We noted that N-3 and O-4 of FdUMP hydrogen bond with the carboxamide group of Asn177 in the TS-FdUMP-PDDF complex (Matthews et aE., 1990). The difference map suggests that this interaction is preserved in the ternary complex with the cofactor as a consequence of co-ordinated movement of Asn177 in response to the change in pucker of the pyrimidine. The conformation of the natural form of CH,-H,PteGlu in solution, R configuration at C-6, has been determined using nuclear magnetic resonance (Poe et al., 1979a)., In particular, these workers have shown that the cofactor’s tetrahydropyrazine ring has a strong preference for the half chair conformation with C-6-H axial, apparently because this conformation partially alleviates strain in the imidazolidine ring (Poe et al., 1979a). On the other hand, 5-formyl, lo-formyl and 5-methyltetrahydrofolate all have C-G-H in an equatorial position (Poe et al., 19796; Poe & Benkovic, 1980) while unsubstituted tetrahydrofolate rapidly exchanges between both conformers (Poe & Hoogsteen, 1978; Furrer et al., 1978). The crystallographic data demonstrate that for the enzyme-bound covalent inhibitory ternary complex, the tetrahydropyrazine ring has its C-6-C-9 bond axial, C-6-H equatorial and that the methylene bridge to C-5 of the nucleotide orients the C-5-F bond equatorial as well. Furthermore, our structural analysis suggests that the folate cofactor and the enzyme’s active site sulfhydryl attack the vinylic carbon atoms of the substrate’s pyrimidine ring from opposite sides, yielding a complex in which the Cys146 bond to C-6 of FdUMP and the C-5-C-l 1 bond are in a trans diaxial relationship. These results are in good agreement with those obtained from 1‘F nuclear magnetic resonance studies of the L. casei TS covalent inhibitory ternary complex (Byrd et al., 1978). Finally, it should be noted that if this stable complex with FdUMP is structurally analogous to that proposed for a true steady-state intermediate in the normal enzymatic reaction, then the ensuing /? elimination across the C-5-C-11 bond should occur with an unusual eclipsed conformation, since the N-5-C-l 1 and C-5-F bonds are approximately syn-periplanar (Fig. 3). At the current resolution and level of crystallographic refinement, we cannot eliminate the possibility that N-5 may actually be slightly gauche to the fluorine, which would be more consistent with the rgF heteronuclear spin-spin coupling data on

Xynthase

941

the L. casei covalent inhibitory ternary complex (Byrd et al., 1978) but does not affect subsequent arguments. 4. Is the Covalent Inhibitory Ternary Complex a Useful Model for the True Catalytic Intermediate? Benkovic and co-workers have shown that the TS-catalyzed reaction proceeds under tight stereochemical control (Tatum et al., 1977). One test for the validity of the covalent inhibitory ternary complex as a model for the true enzyme intermediate would be to use the present results concerning the conformation of the TS-FdUMPCH,-H,PteGlu adduct to predict by analogy the stereochemical course of the one carbon unit transfer and accompanying reduction catalyzed by TX and then to compare this prediction with experimental results. To anticipate the conclusions of such an analysis, we find that syn elimination across the C-5-C-l 1 bond of an enzyme intermediate structurally analogous to the covalent inhibitory ternary complex would generate the wrong chirality at the incipient methyl of dTMP. We then go on to show that a simple conformational isomerization of such an intermediate can lead to anti elimination across the C-5-C-l 1 bond and to subsequent formation of a methyl group with correct chirality. If the covalent inhibitory complex discussed above is truly a stable structural analog of the proposed catalytic steady-state intermediate, then stereochemical arguments based on the known geometry of starting materials, the structure of the intermediate and the enzyme’s mechanism of action should lead to a consistent prediction for the absolute configuration of the chiral methyl produced at the 5 position of dTMP. In what follows we first present a generally accepted minimal mechanism for TS catalysis and then explore the predicted stereochemical course of the reaction in light of what is known about the structures of starting materials and what we now know to be the structure of the covalent inhibitory ternary complex. A wealth of experimental data supports the notion that TS activates dUMP by Michael addition to C-6 of the pyrimidine forming an enolate anion which then attacks the cofactor (Santi & Danenberg, 1984, and references cited therein). Kallen & Jencks (1966) concluded that formation of CH,-H,PteGlu from formaldehyde and H,PteGlu proceeds through an iminium cationic intermediate. Formation of such an intermediate from CH,-H,PteGlu could occur by small torsion angle rotations within the cofactor’s imidazolidine ring that would position the lone pair electrons on N-5 antiperiplanar to the N-l&C-11 bond, thus favoring an antiperiplanar ring opening and creation of an N-5 iminium cation having the Z configuration as shown in Figure 4. Slieker & Benkovic (1984) argued that syn elimination is unlikely owing to conformational restraints of the fused imidazolidine-tetrahydropyrazine ring system. The enzyme-bound

942

D. A. Matthews

Exocycl~c

methylene

Figure 4. Syn elimination from a catalytic covalent intermediate analogous in structure to the covalent inhibitory ternary complex would result in formation of a methyl group with the wrong chirality.

dUMP enolate anion lying below the si face of the activated cofactor is then positioned for nucleophilic attack on C-11 to form the covalent intermediate. One possible conformation for this catalytic intermediate, shown in Figure 4, can be obtained by analogy with the X-ray-derived structure of the covalent inhibitory ternary complex. Succeeding steps in the reaction would then involve elimination across the C-5-C-11 bond to give enzyme-bound 5-CH,dUMP and H,PteGlu followed by transfer of the folate’s C-6 hydrogen to the exocyclic methylene of the uracil ring to form products. If the elimination reaction Gakes place from the syn-periplanar conformation shown in Figure 4, then the resulting exocyclic methylene would have the Z configuration. Since the C-6 hydrogen of H,PteGlu approaches from above the si face of CH,dUMP, the product methyl group would then have stereochemistry R. Experimentally the starting materials indicated in Figure 4 yield a chiral methyl group of configuration S (Slieker & Benkovic, 1984). Thus, a logical gap arises in our efforts to devise a plausible stereochemical hypothesis for the TS-catalyzed reaction based on the structure of the covalent inhibitory ternary complex. An inconsistency between the predicted chirality and that observed experimentally could arise either from erroneous

et al.

assumptions concerning the enzyme’s putative mechanism of action or from being misled by the assumed structural resemblance bet’ween the covalent inhibitory ternary complex and the enzymatitally relevant covalent intermediate. Given the preponderance of evidence consist*ent with t,he general features of the TX mechanism summarized above, we must critically examine the likelihood that a structure analogous to that found for the covalent inhibitory ternary complex funetions as the intermediate that immediately precedes the elimination reaction and leads to formation of the exocyclic methylene in the enzyme-catalyzed reaction. Careful examination of the TS-FdUMP-CH,H,PteGlu structure as a, model for the catalytic intermediate reveals several potential problems. We noted above that in the covalent inhibitory ternary complex the N-5-C-l 1 and C-5-F bonds are nearly syn-periplanar. Syn eliminations are not unknown, although anti-eliminations from lower energy staggered conformations are favored. Moreover, compared to thiols, tertiary amines are extremely poor leaving groups leading to the suspicion that the preferred breakdown of such an intermediate might involve an anti elimination across the C-5-C-6 bond of the uracil ring that would ultimately give back the original starting materials. Finally: the C-5-C- 11 elimination requires concomitant loss of the nucleotide’s C-5 proton. Removal of this proton might be facilita,ted by the presence of a nearby basic amino acid or by some inherent chemical feature of the intermediate itself that would render the C-5 prot,on unusually acidic. Based upon our X-ray structure of the covalent inhibitory ternary complex, arguments in support of either possibility are unconvincing.

onformational Intermediate

Isomerization of the covalent Precedes Beta Elimination

Because the covalent intermediate’s mode of binding to TX is critically important in eventuating the stereospecificity of the overall reaction; we have been led to consider the possibility that the true enzyme-bound covalent intermediate, which directly precedes the eliminat’ion reaction, may in fact be a conformational isomer of the analogous structure observed in our X-ray study of the covalent inhibitory ternary complex. The crystal structure of dihydrouridine (Suck et al., 1972) reveals that there are two molecules of the nucleoside in t’he asymmetric unit, both having half chair conformations with C-5 and C-6 on opposite sides of the least-squares plane through N-l, C-2, N-3 and C-4. It is interesting to note that the dihydrouracil rings of these two crystallographically independent molecules are puckered differently; in one case C-5 lies above the plane and in the other below. This suggests that for dihydrouracil the two modes of puckering may have similar energies. -4s noted above, the bound nucleotide’s C-5 is positioned above the least-squares plane through N-l, C-2, N-3 and C-4 wit,h the C-5-C-11 bond axial.

Mecharkm

of Action for Thymidybte

Ii

Figure 5. Proposed conformational catalytic

covalent

intermediate

isomerization of the preceding /I elimination.

Model building experiments indicate that a pyrimidine ring of opposite pucker (C-5 below the plane with C-5-C-l 1 equatorial) is readily accommodated in the TS active site requiring only small adjustments in the relative juxtaposition of the folate and nucleotide parts of the complex or of the covalent intermediate as a whole relative to nearby protein. properties of such a The stereochemical conformational isomer will now be explored. Structural comparison of the two isomers (Fig. 5) reveals nearly a 180” difference in the torsion angle about the C-5-C-11 bond. This difference has two potentially important consequences. First, the C-11-N-5 and C-5-H bonds of the intermediate are now anti-periplanar and second the bridging methylene group is turned over in the TS active site compared to its position in the covalent inhibitory ternary complex. This intermediate could then undergo anti-periplanar elimination across C-5-C-l 1 to give an exocyclic methylene having the configuration required to yield a methyl group with the correct chirality following hydride transfer from H,PteGlu. In addition, there are two other important structural characteristics that make this conformational isomer an attractive candidate for the true intermediate immediately preceding the /? elimination step in the enzyme-catalyzed reaction. Atoms C-4,

Synthase

O-4, C-5 and C-11 are coplanar and consequently the 4 0x0 group of the nucleotide’s pyrimidine ring could be expected to promote the acidity of the C-5 proton and stabilize the resulting carbanion by electron delocalization. Furthermore, as we shall see later, there are conserved features of the enzyme active site that would serve to further stabilize such structures. ‘The C-6-S and C-5-C-11 bonds of this proposed intermediate are neither syn-periplanar nor anti-periplanar and thus would lock the complex into a conformation from which its direct return to starting materials is stereoelectronically disallowed. Finally, it is noteworthy that the postulated pucker of the uracil ring in the catalytic intermediate is similar to that found experimentally with lgF nuclear magnetic resonance for FdUMP covalently linked to an active site peptide of L. casei TS obtained by protease digestion of the covalent inhibitory ternary complex (James et al., 1976). On the assumption that this peptide was representative of the native ternary complex, these authors suggested that the observed trans and pseudo equatorial configuration across the 5,6 bond must arise from a ring inversion since, on chemical grounds, initial attack on the uracil ring should occur perpendicular to its plane. We have reached a similar conclusion arguing from a completely different set of initial data. Since in the native enzyme the complex between FdUMP and CH,-H,PteGlu does not undergo the isomerization that we postulate for the natural substrate, the conformation having the N-5-C-11 bond of CH,-H,PteGlu syn or slightly gauche to the C-5-F bond of FdUMP must be thermodynamically more stable than the isomer with these two bonds in an anti configuration. In summary, we have argued that given what is known about the TS mechanism and about the absolute stereochemistry of starting materials and products, the enzyme-bound covalent intermediate that immediately precedes the C-5-C-l 1 elimination cannot be structurally analogous to the native covalent inhibitory ternary complex. A readily obtained conformational isomer of this structure does, however, possess all the chemical and geometrical characteristics necessary for facile conversion to end products of correct chirality.

6. Ligand-induced

Conformational

Changes

A qualitative indication of certain conforma,tional changes that occur on ligand binding to TS can be obtained by comparing distance plots for the apo enzyme and ternary complex structures. A particularly useful representation is the difference distance plot in which the matrix element dij represents the distance between a-carbon atoms i andj in the ternary complex structure minus the corresponding distance in the apo enzyme structure. Such a matrix is symmetric; however, it is convenient to display negative values above the diagonal and positive values below. The difference distance plot for all 264 a-carbon atoms in a single subunit of

D. A.

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Matthews

et al.

difference distance plot’ shown in Figure 6. However, analysis of a distance plot for the dimer coupled with least-squares superposition based on atoms in the apo enzyme and ternary complex that do not move indicate that the principal components of this motion involve displacement of helices I, II, VII, VIII and residues 256 to 260 toward 65 to 90 and 133 to 147 on the opposite side of the active site. Thus, we conclude t,hat in addition to movements at the C terminus and in the Arg21 loop, ternary complex formation results in a significant inward displacement of the upper left-hand wall of the TS active site (Fig. 2 of Matthews et al., 1990) so as to provide a tight induced fit for the bound ligands.

240 220 200 180 ISOf 140 120 100 80

60 40 20

m I/,

0

20

,

III

40

80 60

I

120 100

.-

WC. I

I

180

a-

. A Model

II

200

160

140

II

240 220

260

Figure 6. Difference distance piot (covalent inhibitory ternary complex minus apo enzyme) using all a-carbons in a single subunit of E. coli TS. Negative values above the diagonal, positive values below. Contours are at 1 a and 2 a. E. coli TS is shown in Figure 6. It is immediately apparent that ternary complex formation causes a pronounced general tightening of the TX structure as evidenced by the many matrix elements above the diagonal with magnitudes greater than 1 A in contrast to the very few such elements of comparable magnitude below the diagonal. Such changes are consistent with previous hydrodynamic studies of the protein in solution where it was shown that the Stoke’s radius for L. casei TS decreases 3.5% on going from the apo enzyme to the ternary complex with FdUMP and CH,-H,PteGlu (Lockshin & Danenberg, 1980). Most notable are matrix elements 3.5 to 6 i% in magnitude connecting the four carboxy-terminal a-carbon atoms and several residues in the Arg21 loop (1 to 2 A in magnitude) to most other a-carbon atoms in the TS subunit. These large negative difference distances arise from ligand-induced movement of the protein’s C terminus away from its exposed highly solvated position in the apo enzyme into the active site where it covers one face of the bound folate cofactor. There is a co-ordinated movement of the loop connecting P-strand A to B-strand B into a new position where the guanidinium sidechain of Arg21 can now make charge-mediated hydrogen bonds with the C-terminal carboxylate group and a water-mediated hydrogen bond with N-l of the cofactor, thus anchoring residues 261 to 264 across the top of the active site cleft. Smaller movements on the order of 1 to 1.5 A are indicated by matrix elements linking a-carbon atoms for residues 65 to 90 with 1 to 18, 44 to 52, 168 to li5, 208 to 224 and 253 to 260 and also for residues 133 to 147 linked with 168 to 175, 208 to 224 and 256 to 260. The direction of these movements cannot be deduced directly from the subunit

for Thymidylate

Synthase Catalysis

We begin this section with a few generalizations concerning the TS active site. The putative catalytic residues, as distinct from those involved primarily in binding substrate and cofactor, lie on one side of the active site cavity adjacent to N-5, C-6, C-9 and N-10 of CH,-H,PteGlu and C-4, O-4, C-5 and C-6 of the bound nucleotide positioned directly below the cofactor’s pyrazine ring. The single exception is Asp169 located on the opposite side of the binding site adjacent to N-3 and O-4 of the folate molecule. At least eight ordered solvent molecules are buried in the TS active site, several of which, based on their proximity to bound ligands and proposed chemically important residues, could have direct cata.lytic roles, possibly in shuttling protons. A more complete understanding of just how these amino acids participate in the TS-catalyzed reaction will require detailed kinetic and crystallographic analysis of site-directed mutant TS enzymes. Nevertheless, the current structural studies are suggestive with respect to how these functional groups may contribute to catalysis, We have seen that TS catalysis proceeds through a condensation intermediate in which C-5 of the substrate is alkylated by a methylene group linked to N-5 of the folate cofactor. It is well documented that nucleophilic addition of Cys146 to C-6 of dUMP activates the uracil ring by converting the 5 carbon to a powerful nucleophile. Enzyme-catalyzed activation of CH,-H,PteGlu to form the corresponding 5 iminium ion a,pparently precedes attack on dUMP by Cys146 (Santi el al., 1987). How then might TS facilitate these two chemical processes? (a) Activation

of dUMP

The structure of TS is unusual in that the central P-sheet in each subunit, is interrupted by an extended series of stacked P-bulges. At least. eight conserved residues contribute directly to the locai stability of this secondary structural feature which begins on the protein surface and extends down the sheet for a distance of 20 A into the active site where it controls the exact positioning of Cysl46 by hydrogen bonding to the backbone carbonyl oxygen

MechuGm

?f Action .for Thymid$late

atoms of residues 145 and 146 (Matthews et al., 1989). Munroe et al. (1978) studied the kinetics of TS sulfhydryl group modification by various thiolspecific reagents and concluded that the catalytic cysteine has a pK, value of around 8. However, over the pH range 5.8 to 68 they found a rate of inactivation significantly greater than could be accounted for by a simple titration of Cys146. These observations led them to postulate the existence of a general base in the active site of TS that activates the catalytic cysteine residue, perhaps functioning in a manner analogous to a key histidine residue in papain that leads to enhanced nucleophilicity for Cys25. Interestingly there is a histidine residue in the TS active site, His147, adjacent to the uracil ring of bound FdUMP. Could this histidine be responsible for activation of Cys146? We believe activation by general base proton abstraction is unlikely for several reasons. The distance separating Sy of Cys146 and the closest side-chain nitrogen of His147 is 5.7 A in the apo enzyme which is too far for proton transfer. Both residues are similarly positioned in various ternary complexes. In principle this distance could be reduced to 3.5 A by a 120” rotation about the Ca-Cp bond. However, such a side-chain dihedral (g-) is not allowed for cysteine residues in a-helical secondary structure owing to unfavorable steric interactions between Sy and main-chain C and N atoms (McGregor et al., 1987). Local backbone geometries for residues 146 to 147 in TS are approximately a-helical. Other evidence against the direct role of His147 in activating the catalytic cysteine of TS is its substitution by Val in Bacillus subtilis phage 43T TX and the only slightly altered enzyme activity for an engineered mutant of E. coli TS in which His147 is replaced by Asn (J. E. Villafranca, K. Welsh & D. A. Matthews, unpublished results). The catalytic thiol of TS resides approximately 3.6 A from ordered solvent molecule Wat415, the backbone oxygen of Ala144 and a side-chain NH, of invariant Arg166. The presence of a positively charged guanidinium side-chain in close proximity to Sy of Cys146 would be expected to electrostatically stabilize the anionic form of the thiol group in which the proton has been passed off to the adjacent ordered solvent molecule as shown in Figure 7. Some evidence consistent with this mechanism comes from the effect of polyoxyanions in modulating the rate of TS sulfhydryl group modification by methyl methanethiolsulfonate and 5,5’ dithiobis(2-nitrobenzoic acid). Addition of phosphate to inactivations performed in Tris. HCl at pH 7.4 dramatically decreased the rate of inactivation (Lewis et al., 1978). As noted earlier, Arg166 is one of four arginine residues that interacts with the 5’ phosphate of FdUMP (Matthews et al., 1990). According to our model, the presence of bound phosphate proximate to the Arg166 side-chain would tend to counterbalance the arginine-induced electrostatic stabilization of the thiolate anion,

Xytithase

945 H ‘O/”

Wat415 !-I

Pro145 ,/

I

Arg166 Argi26’

\

Figure 7. Hydrogen bonding within the active site of thymidylate synthase orients the guanidinium side-chain of Arg166 close to the side-chain of Cys146 where it can stabilize an anionic form of the thiol group in which the sulfydryl proton is passed off to adjacent Wat415.

thereby reducing the sulfhydryl’s nucleophilicity. It is intriguing to speculate that exact positioning of the substrate’s phosphate group near the guanidinium side-chain of Arg166 may be important in modulating the nucleophilicity of Cys146, thus perhaps influencing both initial attack at C-6 of dUMP and final elimination of Cys146 from the enzyme-dTMP adduct to yield products. Once formed, the resulting enolate anion is stabilized by hydrogen bonding between its 4 oxygen and Wat401. and the carboxamide of Asn177. (b) Activation of 5,10-methylenetetrahydrofolate and

formation

of

the covalent ternary

complex

Because of rigidity in the tetrahydropyrazineimidazolidine fused ring system, CH,-H,PteGlu is a considerably more open, elongated structure as the free molecule than in the covalent inhibitory ternary complex. This is not surprising since cleavage of the C-11-N-10 bond and subsequent alkylation of the nucleotide’s 5 carbon softens torsion angle constraints for rotations about the C-6-C-9 and C-9-N-10 bonds thus permitting the p-aminobenzoylglutamate portion of the molecule to rotate up over the cofactor’s pteridine ring. In order to gain further insights into how the enzyme might facilitate cleavage of the C-l 1-N-10 bond and lead to formation of the N-5 iminium ion, we attempted to dock the solution conformation for CH,-H,PteGlu proposed by Poe et al. (1979a) to TS in such a way that the pteridine ring coincided with its known position in the covalent inhibitory ternary complex. Much to our surprise, we discovered that the solution conformation of CH,-H,PteGlu cannot be accommodated in the TS active site without major protein conformational changes. When a free molecule of CH,-H,PteGlu encounters TS containing bound nucleotide, it has very little freedom with respect to how it can conformationally adjust to steric constraints imposed on it within the TS active site cavity. The

946

D. A. Matthews

only possible torsional adjustments are exactly those that will drive the free cofactor molecule toward the transition state for cleavage of the C-11-K-10 bond. In what follows, we present a structural hypothesis for TS-mediated activation of CH,-H,PteGlu. When free folate cofactor is modeled into the TS active site, the benzoylglutamate portion of the molecule collides with backbone and main-chain atoms in two regions: the amino-terminal six residues of helix II (51 to 56) and residues 77 to 79, comprising part of a loop at one edge of the active site cavity. In order to relieve these energetically unacceptable contacts, the imidazolidine ring must rotate upward, decreasing its angle with respect to the pyrimidine ring from 120” in the free molecule (Poe et al., 1979a) to something less. Such a conformational change will move the cofactor’s benzoylglutamate side-chain over toward the open channel at the entrance to the active site, closer to where this group binds in the covalent inhibitory ternary complex. Although this altered conformation is energetically strained, it has the attractive feature of positioning the C-l 1-N-10 bond more nearly antiperiplanar to the lone pair of electrons on N-5 while partially relieving unfavorable steric contacts with the protein. In the extreme position where the angle between the pyrimidine and imidazolidine rings approaches 90”, the bond to be cleaved and the N-5 lone pair electrons are exactly anti-periplanar, which should result in facile elimination. However, the energetic cost of such an extreme conformational change in this fused ring system probably is prohibitive. Rather, we believe that final repositioning of the benzoylglutamate side-chain to relieve residual unfavorable cont,acts with the protein is of the accomplished by a pyramidylization cofactor’s 10 nitrogen. Examination of small molecule X-ray structures for folate and methotrexate indicates that the 10 nitrogen in these molecules is nearly planar (Mastropaolo et al., 1980; Sutton et al., 1986). A more general survey of conformations found in crystal structures of nitrogen-containing molecules shows that in cases where nitrogen has aromatic substituents, there is a continuous distribution of conformations from that typical of the sp2 to that typical of the sp3 hybridization state (Andrews et al.: 1988). Thus, the energetic costs of this rehybridization at N-10 are probably modest. A partially or fully pyramidylized nitrogen is more basic than its sp2-hybridized counterpart. Enzyme-mediated protonation of such a nitrogen would be expected to facilitate cleavage of the C-11-N-10 bond (Benkovic, 1980) and may be especially important in the TS-catalyzed reaction, since the C-11-N-10 bond of CH,-H,PteGlu probably cannot orient perfectly anti-periplanar to the N-5 lone pair of electrons because of conformational constraints. Our model indicates that such a geometrical distortion of the 10 nitrogen toward sp3 hybridization coupled with modest rotation of the cofactor’s imidazolidine ring above the adjacent tetrahydropyrazine ring not only has the desired effect of

et a.1.

relieving unfavorable steric contacts between the cofactor’s benzoylglutamate side-chain and nearby protein, but significantly, it also points the N-10 lone pair of electrons directly toward a buried conserved glutamic acid residue, Glu58. The earboxyl side-chain of Glu58 is 4 A from N-10 in the covalent inhibitory ternarv complex and is thus ideally positioned to stabilize protonation of t,he cofactor’s 10 nitrogen prior to cleavage of the C-11-N-10 bond either directly or through a nearby fixed solvent molecule. Although there are several such fixed solvent molecules in the vicinity, 61~58 is otherwise completely buried when substrate and cofactor bind to the TS active site, suggesting that it rnay have an elevated pK value and could even be the source of the proton delivered to N-10. To summarize the conclusions of t,his section, we have argued that t,he TS active site has evolved to bind a strained form of CH,-H,PteGlu in which the imidazolidine ring is rotated into a position that orients the C-II-N-10 bond more nearly antiperiplanar to the N-5 lone pair than it is in the free molecule while simultaneously imparting additional SP3 character to the 10 nitrogen. The enhanced basicity of the pyramidylized nitrogen and its close proximity to buried Glu58 results in protonation of N-10 and concomitant facile cleavage of the C-11-N-10 bond leading to formation of the N-5 iminium ion. Attack on dUMP by the sulfhydryl group of Cys146 convert,s the subst,rat,e’s 5 carbon into a powerful nucleophile. The resulting enolate anion is oriented in the TS active site directly underneath CH,-H,PteGlu such that enzyme-catalyzed generation of the 5 iminium ion can lead directly to formation of the covalent intermediate. From arguments presented above, this intermediate must then undergo a conformational isomerization before eliminating tetrahydrofolate and a proton to generate the enzyme-bound exocyclic methylene. Does the TS structure provide any insights into how such an elimination might occur? (c) Breakdown

of the covalent by /3 elimination

intermediate

Since tertiary amines are in general poor leaving groups, enzyme participation may be required for facile breakdown of the covalent intermediate. Tn what follows we argue that’ conserved Asp169 serves as a general acid catalyst for this step in the overall reaction, A potentially key finding in the current study is that a previously unsuspected parallel may exist between certain mechanistic aspects of reactions catalyzed by TS and the other folate-dependent enzyme of known structure, DHFR. DHFR eatalyzes reduction of the K-5-C-6 double bond of H,PteGlu by preprotonation of N-5 followed by hydride transfer from NADPH to the resulting C-6 carbonium ion. On the basis of struct,ural studies of E. coli DHFR some of us argued t,hat an acidic amino acid deep in the enzyme active site (Asp or Glu depending on species) could function as the proton donor (Matthews et al., 1978). Subsequent

Mechanism

of Action for Thymidylate

site-directed mutagenesis experiments confirmed this suggestion (Howell et al., 1986). An observation of particular significance in the current context is that in TS as well there is a conserved Asp similarly positioned with respect to bound folate. Although the DHFR and T&catalyzed reactions overall are dissimilhr, proton donation to the folate’s 5 nitrogen may be a common feature of both. TS-mediated protonation at N-5 of the catalytic covalent ternary complex would convert a tertiary amine into a quaternary amine, thus greatly facilitating the subsequent elimination reaction. Figure 8 indicates how conserved Asp169 in TS is positioned relative to bound H,PteGlu. Note that one of the carboxyl oxygen atoms hydrogen bonds with N-3 of the pteridine. Although N-5 is too distant for direct proton transfer, such a process could be mediated by the intervening lactam of the bound folate. If the lactam were trapped by enzyme-catalyzed protonation of N-3, as shown in Figure 8, then the proton on O-4 would be positioned for transfer to the electron lone pair on N-5 of the covalent intermediate directly or via an intervening water molecule, thereby promoting elimination. Besides its buried location in the TS active site, other evidence consonant with the possible role of Asp169 as a proton donor comes from an examination of its side-chain conformation. As can be seen in Figure 8 the backbone nitrogen of Leu172 and a nearby ordered solvent molecule Wat403, hydrogen bond to one of the Asp169 carboxyl oxygen atoms constraining the side-chain to adopt an unusual conformation in which the putative O-H bond is anti to the C=O bond. Gandour (1981) argued that carboxylate is a weaker base when constrained to accept a proton from the anti direction. It follows that when protonated such a carboxyl should be a stronger acid if proton donation occurs from the anti rather than syn conformation. In this context it is of interest to note that the side-chain of Asp169 is positioned directly underneath the amino terminus of a seven-turn a-helix, VIII. The dipole field from the buried helix should provide considerable stabilization of the developing negative charge on Asp169 during proton translocation. Turning our attention now to the other bond, which must be broken when the covalent intermediate collapses, it has been suggested that a basic amino acid may reside proximate to the C-5 hydrogen of dUMP in the covalent ternary complex and act to catalyze proton abstraction concomitant with elimination to form the exocyclic methylene. Earlier we proposed a structural model for this intermediate based on our experimental structure for the analogous complex with FdUMP and our assertion that the catalytic ternary complex must undergo a conformational isomerization in order to provide a consistent interpretation of experimental results for the overall stereochemistry of the TS-catalyzed reaction. An interesting chemical feature of this proposed intermediate is the expected acidity of the C-5 proton owing to the possibility of extensive electron delocalization in its conjugate base.

Synthase

947

Leu172

Figure 8. Proposed mechanism for breakdown of the catalytic covalent intermediate involves proton transfer from Asp169 to N-5 of tetrahydrofolate and proton abstraction from C-5 of dUMP by WaMOl. Proton donation to the substrate’s 4 0x0 group creates partial carbonium ion character at the incipient methyl of dTMP whieh would facilitate transfer hydride from tetiihydrofolate.

Enzyme-mediated protonation at N-5 of the covalent intermediate by Asp169 and the inherent acidity of the C-5 proton itself may be sufficient to favor deprotonation at C-5 even in the absence of a strong base nearby. This appears to be the case, since the only basic amino acid in this portion of the active site is His147, which resides 4.5 a from fluorine in the covalent inhibitory ternary complex but, based on site-directed mutagenesis studies, is not required for TS catalysis (J. E. Villafranca, K. W&lsh & D. A. Matthews, unpublished results). The C-5 proton of dUMP is most probably transferred directly to WaMOl, which makes van der Waals’ contact with the nucleotide’s fluorine atom and is hydrogen-bonded to His147, Glu58 and the 4 0x0 group of FdUMP. Another potential but less likely acceptor of the C-5 proton, which is released during the elimination reaction is the side-chain hydroxyl of Tyr94, positioned 3.3 a from the fluorine of FdUMP. This hydroxyl is adjacent to but apparently not hydrogen-bonded to WaMOl in the covalent inhibitory ternary complex. If proton translocation from C-5 of dUMP to Wat4Ol occurs concomitant with /? elimination leading to formation of an exocyclic methylene at C-5, then protonated WaMOl would be ideally positioned to favorably influence the next chemical step in the overall reaction, namely hydride transfer from H,PteGlu to the =CH, by protonating the

948

D. A. Matthews

substrate’s 4 0x0 group. The resulting formal carbonium ion at the C-5 methylene should greatly facilitate hydride transfer from H,PteGlu. Recall that in the covalent inhibitory ternary-complex, C-6-H of H,PteGlu is in an equatorial conformation. Once the conformational constraints on H,PteGlu are released concomitant with elimination, the pyrazine ring of H,PteGlu can isomerize to the equally stable conformation with C-6-H axial (Poe & Hoogsteen, 1978; Furrer et al., 1978), thus positioning the transferable hydrogen directly above the nucleotide’s carbonium ion and in line with its vacant p orbital. Following hydride transfer the 5,6 double bond is regenerated by elimination to form enzyme and dTMP.

The TS covalent inhibitory ternary complex is believed to be & stable structural analog of a true catalytic intermediate. Knowledge of its threedimensional structure suggests for the first time how the enzyme may activate dUMP and CH,-H,PteGlu leading to formation of the intermediate and offers additional support for Friedkin’s (1959) original hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the nucleotide and N-5 of the cofactor. When these structural results are correlated with stereochemical information already known about the mechanism of TS (Tatum et aZ., 1977), it is found that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the C-5-C-11 bond. The elimination itself may be catalyzed by proton transfer to the cofactor’s 5 nitrogen from invariant Asp169 buried deep in the TS active site. The juxtaposition of Asp169 and bound tetrahydrofolate in TS is reminiscent

of that

found

in DHFR

where a similarly positioned conserved carboxyl group apparently acts as a general acid catalyst leading to protonation of the corresponding pyrazine ring nitrogen in H,PteGlu. We thank

Dr Paul Bartlet,t

Cisneros, R. J.; Silks, L. A. $ Dunlap, R. B. (1988). Drugs of the Future, 13, 859-881. Friedkin, &I. (1959). In The Kinetics of Cellular Proliferation (Stohlman, F.; ed.), pp. 97-103, Grune & Stratton, Inc., New York. Furrer, H.. Bieri, J. H. & Viscontini, M. (1978). He/v. Chim. Acta, 61, 2744-2751. Gandour, R. D. (1981). Biorg. Chem. 10, 169-176, Houghton, J. A., Weiss, K. D., Williams, L. G.; Torrance, P. M. & Houghton, P. J. (1986). Biochem. Phcwmacol. 35, 1351-1358. Howell; E. E.; Villafranca, J. E., Warren. M. S., Qatley, S. J. & Kraut, J. (1986). Science, 231, 1123-1128. James, T. L., Pagolotti, A. L., Ivanetich, K. M., Wataya, Y., Lam, S. S. M. & Santi, D. V. (1976). Biochem. Biophys. Res. Commun. 72, 404410. Kallen; R. G. & Jencks, W. P. (1966). J. Biol. Chem. 241, 5851-5863.

8. Conclusions

remarkably

et al.

as well as our Agouron

colleaguesin organic chemistry for helpful discussions and Drs Frank and Gladys Maley for providing the clone of E. coli thymidylate synthase. Also we acknowledge technical assistance provided by Chris Mohr, Warren Schoettlin, Eleanor Howland, Carol Booth, Jennifer Sharp and Ellen Moomaw.

Lewis, C. A., Munroe, W. A. & Dunlap, R. B. (1978). Biochemistry, 17, 5382-5387. Lewis, C. A., Ellis, P. D. & Dunlap, R. B. (1981). Biochemistry, 20; 2275-2285. Lockshin, A. 0. & Danenberg, P. V. (1980). Biochemistry, 19, 42444251. Mastropaolo, D.: Camerman, A. & Camerman, PI’. (1980). Science, 210. 334-336. Matthews, D. A., Alden, R. A.; Bolin, J. T., Filman, D. J.; Freer, S. T., Hamlin, R., Hol, W. G. J.: Kisliuk; R. L., Pastore, E. J.; Plante, L. T., Xuong, N.-h. & Kraut, J. (1978). J. BioE. Ghem. 253, 6946-6954. Matthews, D. A., Appelt, K. 8: Oatley, S. J. (1989). J. Mol. Biol. 205, 44%454. Matthews, D. 8., Sppelt, K., Oatley, S. J. & Xuong, Ng. H. (1990). J. Mol. BioZ. 214, 923-936. IlIcGregor, M. J., Islam, S. A. & Sternberg, M. J. F. (1987). J. Mol. Biol. 198, 295-310. Moore, M. A. i Shmed, F. & Dunlap, R. B. ( 1986). Biochemistry, 25, 3311-3317. Munroe, W. A.. Lewis, C. A. & Dunlap, R. B. (1978). Biochem. Biophys. Res. Commun. 80, 355-360. Poe, M. 85 Benkovic, S. J. (1980). Biochemistry; 19, 45784582.

Poe, M. & Hoogsteen,

Andrews, P. R., Munro, S. L. A., Sadek, M. & Wong, M. G. (1988). J. Ghem. Sot. Perkin Trans. II. 711-718. Belfort, M., Maley, G. F. & Maley, F. (1983). Proc. Nat. Acad. Sci., U.S.A. 80, 1858-1861. Benkovic, S. J. (1980). Annu. Rev. Biochem. 49, 227-251. Berger, S. H. & Hakala, M. T. (1984). Mol. Pharmacol. 25, 303-309. Brunger, A. T. (1988). J. Mol. BioE. 203, 803-816. Byrd, R. A., Dawson, W. H., Ellis, P. D. & Dunlap, R. B. (1978). J. Amer. Chem. Sot. 100, 7478-7486. Edited

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Chem. 253,

Poe, M.? Jackman, L. M. & Benkovic, S. J. (1979a). Biochemistry, 18, 55276530. Poe, M., Hensens, 0. D. & Hoogsteen, K. (19796). J. Biol. Chem. 254, 10881-10884. Santi, D. V. & Danenberg, P. V. (1984). In Folates in Pyrimidine Nucleotide Biosynthesis (Blakeley, R. L. & Benkovic, S. J., eds), pp. 350-398, John Wiley & Sons, New York. Santi, D. V., McHenry, C. S., Raines, R. T. & Ivanetich, K. M. (1987). Biochemistry, 26, 8606-8613. Slieker, L. J. & Benkovic, S. J. (1984). J. Amer. Chem. Sot.

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