ABSTRACT. Atomic structures of thymidylate synthase. (TS) reveal key steps in a multi-step reaction and show quantitatively how conformation change.
Stereochemistry reaction: ROBERT Department 94143-0448,
of a mu1tisep/bipartite thymidylate
M. STROUD
AND JANET
of Biochemistry USA
S. FflER-MOORE
and Biophysics,
University
J.
Key Wordr: site-directed mutagenesis structure enzyme mechanism #{149} dCMP methylase conformalion change polygiutarnale drug target #{149} methyl transfer atomic structure
A DRAMATIC STRUCTURAL CHANCE IS the key to the mechanism of thymidylate synthase (TS,’ EC 2.1.1.45), an essential enzyme for de novo synthesis of dTMP. This change is required to bind and correctly orient the substrate dUMP and cofactor CH2H4folate during the multistep reaction pathway (1). Using derivatives of the substrate and cofactor, and protein altered by mutations, it is possible to go beyond this twostate situation and map intermediate stages of the reaction chemistry. TS is one of the most highly conserved enzymes (2), with a unit evolutionary period of -23 million years - the mean time it took to accept a single amino acid change during the divergent evolution of TS. To date, sequences are known from more than 17 different species of TS. These include the human enzyme, which is a drug target for anti-cancer agents, thymidylate synthases from protozoa, which are generally bifunetional (3), with TS and dihydrofolate reductase present in a single protein, and TS from bacterial and viral species. TS is an essential enzyme to almost all living species and is coded for within many viruses, presumably to support an increased need for DNA synthesis during the viral life cycle. There are only a few organisms that adequately scavenge thymidine from their environment and do not require TS (R. Reid, personal communication). The essential requirement for TS activity makes TS an important drug target for the development of anti-parasitic, antifungal, and possibly anti-viral agents. The many TS species provide an ideal test bed for the design of species-specific inhibitors. The essential nature of TS for DNA synthesis has also led to a powerful genetic complementation assay for active vs. inactive mutants of TS using a synthetic TS gene carried on a high-expression plasmid pSCTS9 (4) in a Thy strain of Escherichia coli (x2913 originally prepared by Russell Thompson) (5). Interesting mutants have been further characterized kinetically, and in some cases structurally. The combination
© FASEB
transfer
synthase
ABSTRACT Atomic structures of thymidylate synthase (TS) reveal key steps in a multi-step reaction and show quantitatively how conformation change is involved in mediating the methyl transfer reaction catalyzed by TS. Numerous alterations in TS produced by mutation, screened by complementation, and further characterized can be understood in terms of the structure and profound structure change required during the TS reaction.Stroud, R. M., Finer-Moore, S. Stereochemistry of a multistep-bipartite methyl transfer reaction: thymidylate synthase. FASEBJ. 7: 671-677; 1993.
0892-6638/93/0007-06711$01.50.
methyl
of California
at San Francisco,
San Francisco,
California
of a high-level bacterial expression system, with an optimized synthetic gene engineered for convenient cassette mutagenesis by having unique restriction sites about equally spaced throughout the entire gene, provides an especially powerful tool for molecular engineering.
METHYL DISCRETE
TRANSFER STEPS
INVOLVES
SEVERAL
The source of the methyl group for transfer to dUMP, the cofactor CH2H4folate, plays a dual role. First, it is a onecarbon donor, and then reductant of the transferred methylene at different steps in the reaction. The major chemical steps in the methylation reaction have been deduced using chemical models as well as by studying the structures and kinetics of the formation of reaction intermediates (6) (Fig. 1). Cys-198[146]2 activates dUMP by covalent attachment to C-6 of the pyrimidine. After CH2H4folate binds, its imidazolidine ring opens, giving a reactive iminium ion that condenses with the activated C-5 of dUMP. Breakdown of the resulting steady-state intermediate (II), including hydride transfer from cofactor to the transferred methylene, generates dTMP and dihydrofolate. The ternary complex of TS, CH2H4folate and the 5-fluoro derivative of the substrate dUMP (FdUMP) have been particularly useful for understanding the enzyme mechanism. This complex is an analog of the steady-state intermediate II of Fig. 1, differing only by substitution of a fluorine for a hydrogen atom at C-5 of dUMP. The reversible, covalent TS. FdUMP . CH2-H4folate complex provides a means of determining the kinetics of the first half of the reaction in detail because the mechanism of formation of this analog complex is thought to be very similar to the mechanism of formation of 11(7).
STRUCTURAL Until recently catalysis. The
STEPS
IN THE
REACTION
PATH
little was known about the enzyme’s role in first crystal structure of TS, that of the Lac-
‘Abbreviations: 5-monophosphate; drofolate; dTMP, drofolate; CB3717, 2 ‘-deoxyuridine
TS, thymidylate synthase; dUMP, 2-deoxyuridine CH2H4folate, 5,lO-methylene-S,6,7,8-tetrahythymidine 5-monophosphate; H2folate, 7,8-dihylO-propargyl-5,8-dideazafolate; FdUMP, 5-fluoro5 -monophosphate; P., inorganic phosphate; V316Am, mutantL. casei 1’S lacking a COOH-terminal valine; PABA, p-aminobenzoic acid. 2Residue numbers refer to the L. casei sequence and are followed by the E. coli 1’S numbering in brackets. Residues from the “second” monomer which enter into discussion of the first are indicated with a prime
(). 671
Two active monomers
sites
formed
by the interface
between
1H2
NC
H2
0 H
I H NH H
O-N’S-EflZ
dbc
nbc..
ring
H2
H4 blat.
00 HNL9CH2 ONEnZ
TS is an obligate dimer of identical subunits that are related by a twofold axis of rotation (Fig. 2). The monomers have been separated only by unfolding the protein. However, catalytically inactive mutant homo-dimers can be unfolded and then refolded to form an active hybrid dimer, suggesting a separate monomer to dimer association step in the folding pathway (17). The structure of TS shows why it is an obligate dimer, as components of each monomer contribute to each of two active sites. There is no conclusive evidence for cooperativity between monomers in the dimer. However, as ligand binding induces large conformational changes that extend throughout the protein, the ligand affinities will depend on the ligated state at both active sites. The dimer interface is formed by a back-to-back appositionof two six-stranded /3-sheets with a unique right-handed twistof one sheetrelative to the other (8). Three contiguous /3-strands bend sharply away from the remainder of the sheet in a “/3-kink” and form one wall of the large active site cavity. The cavity is lined with many conserved side chains, includ#{149})Base ing the active site sulfhydryl, Cys-198[146]. Two layers of helices and loops pack on top of the central /3-sheet. The ordered function
nbc..
When H
Eiiz H nibosi
HI
nibos.
Figure 1. Proposed chemical mechanism (6). The stereochemistry of intermediate crystal structure of E. coli TS #{149} dUMP
Vol. 7
May 1993
monoglutamylated
of binding
cofactor
is not
is used
necessary
there
for
is evidence
that the ligands bind in an ordered fashion in which dUMP binds first, followed by cofactor, and that cofactor leaves before dTMP in a bi-bi reaction sequence (18, 19). These results may be explained in part by the fact that dUMP is an important binding surface for the pterin (or quinazoline)
nba..
for thymidylate II is deduced #{149} CB3717
synthase from the
(12).
tobacillus casel enzyme (8), revealed structural details of the ligand binding site. Subsequently, crystallographic studies of complexes of TS that are analogs of reaction intermediates have provided a window on the reaction, tracing changes in protein environment, at an atomic level, that are required to mediate the chemical steps outlined in Fig. 1. Refined TS crystal structures include unliganded (P,-bound) TS from L. casel (9), E. coli. (2, 10), phage T4 (unpublished), and human species (unpublished), binary complexes of TS with dUMP (9) and with a polyglutamylated cofactor analog (11), ternary complexes that are analogs of the steady-state intermediate (II of Fig. 1) (10, 12-14), and a product complex of TS with dTMP and dihydrofolate (14a). 10-Propargyl-5,8-dideazafolate (CB3717), a 10-nm antifolate designed a potential anti-cancer drug (15), is used in many of the complexes studied by crystallography as it prevents any reaction beyond enzyme with dUMP. It has less internal constraint than CH2H4folate because the imidazolidine ring is opened, and it has a propargyl substituent at N-b. Like CH2H4folate, it induces covalent bond formation between enzyme and dUMP (16); however, CB3717 cannot form a covalent bond to dUMP analogous to the covalent bond between cofactor and dUMP in intermediate II (Fig. 1). CB3717 induces the same conformational change in E. coil TS as does CH2H4folate and binds in a similar orientation as the cofactor (10, 14a).
672
mechanism
ring: 9 of 12 atoms in the quinazoline ring of CB3717 are within 3.8 of some atom of dUMP (12). Galivan et al. (20) show that folates that exhibit no detectable binding without nucleotide bind tightly to the enzyme-nucleotide complex. The intracellular pool of folates consists mainly of polyglutamylated molecules. Polyglutamyl folates have affinities for TS that are in some cases more than 400-fold greater than that of the monoglutamyl form (21). Under certain reaction conditions, the increased binding affinity of polyglutamyl folates for TS results in random binding order of ligands (22). Both monoglutamyl CB3717 and polyglutamyl CB3717 crystallize in complex with E. coli TS, demonstrating that a close analog of the cofactor can bind in the absence of dUMP (11). The quinazoline ring of CB3717 in crystal structures of these complexes is highly disordered, as expected given that dUMP is a major binding determinant of folates. Although the quinazoline ring impinges on the dUMP binding site, its large thermal vibrations indicate it could easily move out of the way to allow dUMP binding and formation of a ternary complex. Thus the structure is consistent with the observation of random binding of ligands under certain reaction conditions.
A
Three-dimensional structure iminium ion intermediate
proves
the
formation
of an
It has long been accepted that an activated form of the cofactor, most likely the iminium ion 5-CH2’H4folate, condenses with dUMP to form the steady-state intermediate (23). Crystals of the dead L. card mutant V3I6Am in a ternary complex with FdUMP and CH2H4folate have trapped the hydroxylated form of this iminium ion (24). The crystal structure clearly shows that the imidazolidine ring of CH2H4folate is
The FASEBJournal
STROUD
AND
FINER-MOORE
Sequestration of ligands cofactor binding
from
solvent
is triggered
by
The structures of native, unliganded TS from L. casei, E. coil phage T4 and human present large cavernous openings that close down progressively as ligands bind. In the binary complexes formed as the substrate dUMP binds, the active sites remain open and very close to the unliganded state (9). On the other hand, the binding of CB3717 by itself produces the closure of the active site, which serves to sequester the reactants from solution (11). These results demonstrate that structure change during catalysis is triggered mainly by the binding of cofaetor, and not by dUMP. The earlier evidence for a conformational change upon ligation with both substrate and cofactor was clear. On complexation, aromatic groups on the protein and of the folate chromophore were perturbed, as detected by Raman spectroscopy, circular diehroism, ultraviolet speetroscopy, and fluorescent spectroscopy (6). In both human and L. casei TS, the Stokes radius decreased by 3.5% upon formation of the TS. FdUMP. CH2H4folate complex (25). The dramatic changes in the spectra and hydrodynamic behavior of L. casei TS on formation of ternary complexes suggest large conformational differences between unliganded TS and TS in ternary complexes with substrate and cofactor analogs. In fact, the differences between P,-bound E. coli TS and TS . dUMP . CB3717, although quite extensive, are not particularly large (12). Segments of helices, /3-sheet, and loops move toward the active site, usually by less than I A, to accommodate ligand binding. The ligand binding side chains do not themselves change conformation as the active site closes. With two exceptions, they are well-ordered and correctly oriented for ligand binding in the P1-bound structures. The greatest segmental shift is in residues 312-316 [260-264] at the COOH terminus. These residues become more ordered in the ternary complex and move more than 4.0 into the active site, forming an important binding determinant for the cofactor.
A
Reorientation
Figure 2. A) Ribbon drawing of the E. coil 1’S dimer with dTMP (in yellow) and H2folate (in green) bound in the active site (l4a). The monomers are different shades of purple. Side chains within 4 A ofthe ligands are displayed in red. The polyglutamyl moiety on H2folate was derived from the complex of E. coii TS with dUMP and tetraglutamyl CB3717 (14). B) Space-filling model of the complex shown in (a) using the same color scheme. Ligands are almost completely sequestered from solvent: only the PABA and polyglu
moietiesof H2folate
are visible
in this
view.
STRUCTURAL MECHANISM OF THYMIDYLATE
for methylene
transfer
dUMP binds to the protein, with its pyrimidine ring in the anti orientation (x’ 170#{176}) and its ribose ring in a C2 endo conformation, and it maintains this conformation on folate binding (9). The pterin ring of CH2H4folate or its analog, the quinazoline ring of CB3717, then binds with its face parallel to the face of dUMP. Folate binding triggers the protein conformational change and displaces several ordered water molecules from the active site cavity of the TS . dUMP binary complex (9). Folate is proposed to bind in two steps to TS . dUMP complexes, first binding in a loose noncovalent adsorption complex, then forming a tight, covalent complex (7). The protein conformational change coincides with the second step and may be essential for properly orienting the pterin ring. For example, deletion of the COOH-terminal residue of L. casel TS interferes with this conformational change (26), and the crystal structure of the V3b6Am mutant in complex with FdUMP and CH2H4folate shows FdUMP and the PABA moiety of the cofactor bound close to their usual binding sites, but the ptenfl ring bound outside the active site cavity (24). Reorientation
opened. The structure also demonstrates that the iminium ion forms early in the reaction, because as a result of the mutation neither the conformational change nor covalent bond formation to substrate has occurred.
of reactants
of reactants
for hydride
transfer
After formation of the covalent steady-state intermediate (II of Fig. 1), H4folate is eliminated to give H4folate and an exocyclic methylene derivative of dUMP (intermediate III of Fig. b). Then a hydride from C-6 of the folate is stereo-
673
specifically transferred to the exocylclic methylene of the dUMP derivative (27). In models of the steady-state intermediate based on ternary complex crystal structures, the C-6 hydrogen of cofactor is poised to protonate the wrong face of the exocyclic methylene to account for the observed stereospecificity (1, 10). However, correct stereospecificity of hydride transfer will occur if the covalent ternary complex undergoes a conformational change that moves the methylene bridge from an axial to an equatorial orientation before elimination of H4folate (10). This conformational change would facilitate loss of the C-S hydrogen by positioning the C-H bond nearly perpendicular to the plane of the pyrimidine ring, so that the incipient negative charge would be in conjugation with dUMP 0-4 (10). Alternatively, the pyrimidine ring of intermediate III must rotate after elimination of H4folate to allow hydride transfer to the correct face of the methylene. The spacious active site cavity can accommodate such reorientation of ligands. Enthalpy release
of water
hydrogen
bonding
drives
product
The product of the reaction, dTMP, binds three to seven times less tightly than the substrate (20, 28, 29). The crystal structure of the E. coli TS product complex, TS . dTMP H2folate, suggests that a conserved ordered water molecule near C-S of dUMP may be responsible for the decrease in binding affinity for dTMP (14a). In binary and ternary complexes of TS, with dUMP, this water is well-ordered and hydrogen bonded to the hydroxyl oxygen of the conserved residue Tyr-146 and to the main chain carbonyl of residue 196. In the product complex it is shifted by the C-7 methyl of dTMP, is only partially occupied, and is not hydrogen bonded to the protein. Thus displacement of an ordered water by methylation of dUMP increases entropy but costs the enthalpic energy of two hydrogen bonds. The net effect may be destabilization of TS #{149} dTMP with respect to TS dUMP.
LIGAND BINDING AND FUNCTIONAL
ROLES OF DISCRETE GROUPS ON THE
Figure
WATERS PROTEIN
3 summarizes the specific contacts between ligands and protein in the covalent ternary complex (1, 12). Four highly conserved arginines, two from each monomer, provide a favorable, positively charged binding surface for the phosphate anion. These four arginines, Arg-23 [21], Arg-218 [166], Arg-178’ [126’], and Arg-179’ [127’], as well as Ser-219 [167] are involved in an extensive hydrogen bonding network around the phosphate. Each arginine forms at least one hydrogen bond to the phosphate oxygens. Arg-23 [21] is also hydrogen bonded to the COOH terminus of the protein. Arg-218 [166] is hydrogen bonded to the carbonyls of residues Pro-196 [144] and Arg-178’ [126’]. Arg-178’ [126’] is hydrogen bonded to the hydroxyl of Tyr-261 [209]. Invariant Tyr-261 [209] and His-259 [207] are the primary contacts to the ribose ring of dUMP: they are hydrogen bonded to 0-3’ on the ribose ring. Hydrogen bonds between the pyrimidine of dUMP and invariant Asn-229 [177] and the main chain amide of Asp 221 [169] are the only other hydrogen bonded contacts between dUMP and the protein. Asn-229 [177] is hydrogen bonded to both 0-4 and N-3 of dUMP. The only protein side chain to hydrogen bond to the cofactor is that of invariant Asp-221 [169]. Other hydrogen bonds involving the cofactor are made either with water or back-
674
Vol. 7
May 1993
bone atoms. The exocylic amino group on the pterin ring is hydrogen bonded to the terminal carbonyl (residue 31S [263]) in the protein. The PABA ring lies in a hydrophobic pocket surrounded by the side chains of the highly conserved residues Ile-81 [79], Leu-224 [172], Phe-228 [176], and Val-314 [262]. The pocket is formed in part by movement of the COOH terminus on ligand binding. Phe-228 [176] and Leu-224 [172] are within van der Waals contact of the PABA ring. Trp-85 [83] and Trp-82 [80] are just below the PABA ring, stacked against the pterin ring. The cofactor is normally linked to a linear sequence of as many as seven glutamate residues that are y-linked or alinked to the PABA ring and to each other (31). Glutamate residues on the cofactor contribute as much as 3.7 kilocalories/mol to the interaction between the cofactor, substrate, and enzyme. The crystal structure of a tetraglutamyl cofactor analog bound in ternary complex with deoxyuridine monophosphate (dUMP) and E. coli TS reveals that interactions between protein and polyglutamyl moiety are primarily electrostatic (14). The only three clearly discernible hydrogen bonds between the tetraglutamyl group and the protein are mediated by ordered water. The tetraglutamyl moiety is not rigidly fixed by its interaction with the protein except for the first glutamate residue nearest the PABA ring of folate. The second, third, and fourth glutamate residues are progressively more disordered and lie in a shallow hydrophilic cleft on the protein surface.
TESTING FUNCTION THE LAST STRAW?
OF RESIDUES
BY MUTATION:
Mutations were introduced into the TS gene, both using replacement sets created by inserting eodons with each possible base at positions one and two, and G or C at the third position (5), and using a series of amber tRNA suppressors and genetic complementation (32, 33). Thus many of the conserved residues, and residues that seem to play a role in the function or binding of reaction components, have been altered for many alternatives. The results emphasize that amino acid conservation is not simply explained. Indeed there may be other as yet unrecognized factors in the evolution of TS, such as its ability to remain insensitive to the binding of unwanted components inside the cell, or interactions between TS and other enzymes. TS remains functional at the level of approximately 1-100% after many of the substitutions, and therefore it can accommodate many different single insults without total inactivation. Several of these together may reduce activity to zero, as it takes many straws to break the camel’s back. The effects of site-directed mutagenesis of some of the more sensitive residues are discussed in relation to the liganded TS structures. The reactive by essential
nucleophile, Arg-218
Cys
198,
is polarized
toward
S
Invariant Cys-198[146], the nucleophile that attacks C-6 of dUMP, activates C-5 of dUMP for condensation with the cofactor (6). Any mutation of Cys-198[146], with the possible exception of C198[146]S, inactivates TS (5, 32, 34, 35). Arg-218[166 cannot be mutated to any of the other amino acids without loss of function (5, 32); therefore it may be considered essential for efficient catalysis by TS. It may function to activate Cys-198[146] through charge interactions or hydrogen bonding with the catalytic sulfhydryl (8). In TS dUMP, the guanidinium of Arg-218 [166] is 3.5 A from
The FASEBJournal
STROUD AND FINER-MOORE
Figure
3. A) Divergent
stereo drawing (30) of the E. coli 1’S active site showing dUMP covalently bound to Cys-198 and the CH2H,folate Important ligand binding residues are labeled with L. casei 1’S sequence numbers and hydrogen bonds are indicated by dashed lines. Waters are shown as black spheres and labeled “Wa?’ Water 7, shown near C-5 of dUMP, may discriminate between dUMP and dTMP binding and could also accept the dUMP C-5 hydrogen during breakdown of intermediate II. B) Same view of the active site cavity shown in a but with CH2H4folate and the upper protein layer cut away to show only the interactions between dUMP and the in the imino
ion form.
protein.
Cys-198 [146], too far to form a good hydrogen bond to the SH group but near enough that the charge on Arg-218[166] could stabilize the more nucleophilic thiolate anion S-form of Cys-198[146] and lower the pKa of Cys-198[146].
Four arginines bind phosphate with up to eight hydrogen b d on s Four positively charged arginine side chains bind to the phosphate on dUMP, or to P1, which acts as a weak inhibiSTRUCTURAL
MECHANISM
OF THYMIDYI.ATE
tor of TS. Each offers electrostatic and hydrogen bonding stabilization. TS is relatively insensitive to substitution by mutation of invariant residue Arg-179’ [127’] to several other residues in L. caseii [36] or E. coli TS [32]. All mutants are active; the effects of substitutions are primarily on the association rate of dUMP (36). Thus Arg-179’[127’] plays a role in .
.
.
.
.
.
dUMP binding, but is not an essential binding determinant. Arg-178’[126’] is more sensitive to substitution, but several mutants at this position are active (5, 32, 35). Any mutation of Arg-23[21] severely compromises enzyme activity (S, 32, 675
37). Besides being hydrogen bonded to the phosphate in ternary complexes of E. coil TS, Arg-178’[126] and Arg-23[21] are also hydrogen bonded to Tyr-261[209] Oi and the earboxyl terminus of the protein, respectively. These arginines may have multiple roles in stabilizing the ligand binding sites and the closed conformation of the protein. Changing
Asn
229
to Asp
makes
1’S a dCMP
methylase
Asparagine 229[177] makes two of the three hydrogen bonds from the protein to the pyrimidine of dUMP. Although this residue seems to be an important binding determinant for dUMP, its more important function seems to be ensuring specificity for dUMP binding. When Asn-229[177] of L. casei TS is substituted with the hydrophobic residues Cys, lie, Met, and Leu, all are active (38). When Asp is substituted for Asn in either L. casel (38) or E. coil TS (39), kcat is drastically reduced; but in contrast to wild-type TS, these mutants bind dCMP and catalyze its methylation. Based on kcat/Km values, the mutant has a 40-fold specificity for dCMP methylation over dUMP methylation (38). The
essential
COOH
terminus
closes
the
gate
Of 19 possible substitutions for the terminal valine of L. cosel TS, 18 were active by an in vitro assay (40). Alterations increased Km for folate and decreased kcat, but had little effect on Km for dUMP. Hydrophobic substitutions were best tolerated. When the COOH-terminal residue is eliminated completely by introduction of the amber mutation, the protein no longer catalyzes dTMP formation (28). It does, however, catalyze partial reactions, such as thiol-dependent dehalogenation of 5-bromo-dUMP, which rely on activation by Cys-198[146] but do not require folate. Affinity of the amber mutant TS #{149} FdUMP for CH2H4folate and the rate of formation of the covalent ternary complex are much reduced. These results are consistent with the structural results that the COOH-terminal earboxyl, and not the side chain, is involved in folate binding and stabilization of the closed protein conformation. Essential bulkheads stabilization?
for binding
or for transition
state
Nearly all mutations of Tyr-261[209] (5), Trp-82[80] (32), or Asp-221[169] (32) are inactive. The obvious role of these residues is in binding ligands (see Fig. 3). However, other important ligand binding residues, such as Asn 229[177], are relatively insensitive to mutation. More thorough study may reveal whether these residues have an additional role in catalysis. The
essential
roles
of water
molecules
in catalysis
Breakdown of the covalent ternary complex (II of Fig. 1) requires loss of the C-5 proton of the pyrimidine (C-5H). C-5H is poorly acidic, expected to have a high pKa, and to leave only with assistance from a catalytic base within the enzyme. However, many substitutions of His-199[147], Asn-229[177], and Tyr 146[94] in the vicinity of C-5H and 0-4 of the pyrimidine ring can be made without loss of activity (5, 32, 35, 41). Thus there may be no single residue that serves to abstract C-5H. In addition, the pKa of a solvent accessible imidazole side chain of His is normally about 7.0, far from the pKa of the C-5H. However, conserved residues and ordered water molecules that form a network of hydrogen bonds in a cavity directly above the pyrimidine ring may jointly lower the pKa of C-5H by stabilizing the incipient enolate. In particular, water Wal of Fig. 3, which is
676
Vol. 7
May 1993
directly hydrogen bonded to 0-4 of dUMP, is conserved in ternary and binary complexes of TS (9, 12-14) and could be essential for stabilizing the enolate intermediate (1). The pKa of this water may be finely tuned by the hydrogen bonding network to maximally lower the energy of the transition state. The conserved water near C-5 of dUMP (water Wa7 of Fig. 3) is then the only moiety ideally positioned to receive the now more acidic C-5 proton (14a). Wa7 may also function to help repel the product dTMP after methylation at the nearby C-5 with respect to the binding of substrate dUMP.
CONCLUSION Methyl transfer by TS involves many steps. Crystallography shows that the cofactor opens to form an iminium ion soon after binding even before the major conformational change takes place. The active sites of the obligate dimer are formed at the interface between subunits in cavernous clefts that close down on the reactive components, yet provide the framework for many very well-ordered water molecules in the substrate-cofactor-enzyme complexes. About 25 side chains form each active site, and 19 of them are totally conserved. Most of the conserved side chains can be substituted for others by mutagenesis with only impairment of function. However, at least six residues are especially sensitive to substitution. These are the reactive nucleophile Cys-198[146], nearby Arg-218[166] that may serve to polarize the nucleophile, Arg-23[21], Tyr 261[209], Trp-82[80], Asp-221[169]. No group on the protein seems close enough to C-5H to act directly as an acid/base catalyst for facilitated proton transfer. Highly ordered water molecules, structured within the active site, presumably assist in mediating both C-5H proton transfer and transition state stabilization. Figure 2 was made by Julie Newdoll using the Midas Plus program written by Conrad Huang, Eric Pettersen, and Greg Couch at the UCSF Computer Graphics Laboratory. Research was supported by National Institutes of Health grants RO1-CA-41323 to J.F.M. and R.M.S. and GM24485 to R.M.S.
REFERENCES 1. Finer-Moore, J. S., Montfort, W. R., and Stroud, R. (1990) Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase. Biochemistry 29, 6977-6986 2. Perr#{231} K. M., Fauman, E. B., Finer-Moore, J.S., Montfort, W R., Maley, G. E, Maley, E, and Stroud, R. M. (1990) Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases. Proteins: Struct. Funct. Genet. 8, 315-333 3. Ivanetich, K. M., and Santi, D. V. (1990) Thymidylate synthase - dihydrofolate reductase in protozoa. Exp. Parasitol. 70, 367-371 4. Climie, S., and Santi, D. V. (1990) Chemical synthesis of the thymidylate synthase gene. Proc. NatL Acani Sci. USA 87, 633-637 5. Climie, S., Ruiz-Perez, L., Gonzalez-Pacanowska, D., Prapunwattana, P., Cho, S.-W., Stroud, R., and Sand, D. V. (1990) Saturation site-directed mutagenesis of thymidylate synthase. j Biol. Chem. 265, 18776-18779 6. Sand, D. V., and Danenberg, P. V. (1984) Folates in pyrimidine nucleotide biosynthesis. In Foiates &Pterins: Vol. 1- Chemistry and Biochemistry of Folates (Blaldey, R. L., and Benkovic, S. J., eds) pp. 345-398, John Wiley and Sons, New York
The FASEB journal
STROUD AND FINER-MOORE
7. Santi, D. V., McHenry, C. S., Raines, R. T., and Ivanetich, K. M. (1987) Kinetics and thermodynamics of the interaction of 5-fluoro-2-deoxyuridylate with thymidylate synthase. Biochemistry 26, 8606-8613 8. Hardy, L. W, Finer-Moore, J.S., Montfort, W. R., Jones, M. 0., Santi, D. V., and Stroud, R. M. (1987) Atomic structure of thymidylate synthase: target for rational drug design. Science 235, 448-455 9. Finer-Moore,J.,
Fauman,
E. B., Foster,
P. C., Perry,
K. M., and
Stroud, R. M. (1993) Refined structures of substrate-bound and phosphate-bound thymidylate synthase from Lactobacillus casei. j Mol. Biol. In press 10. Matthews, D. A., Villafranca, J. E., Janson, C. A., Smith, W. W, Welsh, K., and Freer, S. (1990) Stereochemical mechanism of action for thymidylate synthase based on the x-ray structure of the covalent inhibitory ternary complex with S-fluoro-2’ -deoxyuridylate and S,10-methylenetetrahydrofolate. j Mol. Biol. 214, 937-948
11. Kamb, A., Finer-Moore,J. S., and Stroud, R. M. (1992) Cofactor triggers the conformational change in thymidylate synthase: implications for an ordered binding mechanism. Biochemistry 31, 12876-12884 12. Montfort, W. R., Perry, K. M., Fauman, E. B., Finer-Moore, J. S., Maley, G. F., Hardy, L., Maley, F., and Stroud, R. M. (1990) Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate. Biochemistry 29, 6964-697 7 13. Matthews, D. A., Appelt, K., Oatley, S. J., and Xuong, N. H. (1990) Crystal structure of Escherichia coii thymidylate synthase containing bound 5-fluoro-2’-deoxyuridylate and lO-propargyl5,8-dideazafolate. ,J. Mol. Biol. 214, 923-936 14. Kamb, A., Finer-Moore,J. S., Calvert, A. H., and Stroud, R. M. (1992) Structural basis for recognition of polyglutamyl folates by thymidylate synthase. Biochemistry 31, 9883-9890 l4aFauman, E. B., Rutenber, E. E., Maley, E, and Stroud, R. M. (1993) Water-mediated substrate/product discrimination: the product complex of thymidylate synthase at 183 A. Biochemistry In press 15. Jones, T R., Calvert, A. H., Jackman, A. L., Brown, S. J., Jones,
16.
17.
18.
19.
M.,
and
Harrap,
K.
R.
(1981)
A potent
on
the
dissociation
of
S-fluoro-2-deoxyuridylate from thymidylate synthetase: evidence for an ordered mechanism. Biochemistry 17, 4018-4024 20. Galivan, J.H., Maley, G. F., and Maley, F. (1976) Factors affecting substrate binding in Lactobacilius casei thymidylate synthetase as studied by equilibrium dialysis. Biochemistry 15, 356-362 21. Lu, Y.-Z., Aiello, P. D., and Matthews, R. G. (1984) Studies on the polyglutamate specificity of thymidylate synthase from fetal pig liver. Biochemistry 23, 6870-6876 22. Ghose, C., Oleinick, R., Matthews, R. G., and Dunlap, R. B. (1990) Kinetic studies on thymidylate synthase from Lactobacilius casez In Chemistry and Biology of Pt#{128}ridines 1989 (Curtius, H.-Ch., Ghisla, S., and Blau, N., eds) pp. 860-865, Walter de Gruyter
STRUCTURAL
MECHANISM
OF THYMIDYLATE
28. Daron,
29.
30.
31.
32.
33.
34.
antitumour
quinazoline inhibitor of thymidylate synthetase: synthesis, biological properties, and therapeutic results in mice. Eur. J. Cancer 17, 11-19 Pogolotti, A. L., Jr., Danenberg, P. V., and Santi, D. V. (1986) Kinetics and mechanism of interaction of 10-propargyl-S,8dideazafolate with thymidylate synthase. J. Med. Chem. 29, 478-482 Perry, K. M., Pookanjanatavip, M., Zhao, J.,Santi, D. V., and Stroud, R. M. (1992) Reversible dissociation and unfolding of the dimeric protein thymidylate synthase. Protein Sci. 1, 796-800 Lorenson, M. Y., Maley, G. F., and Maley, F. (1967) The purification and properties of thymidylate synthetase from chick embryo extracts. j Biol. Chem. 242, 3332-3344 Danenberg, P. V., and Danenberg, K. D. (1978) Effect of 5,10-methylenetetrahydrofolate
and Company, Berlin 23. Benkovic, S. J.,and Bullard, W. P. (1973) On the mechanisms of action of folic acid cofactors. Prog. Biorg. Chem. 2, 133-175 24. Perry, K. M., Carreras, C. W, Chang, L. C., Santi, D. V., and Stroud, R. M. (1993) Structures of thymidylate synthase with a C-terminal deletion: role of the C-terminus in alignment of dUMP and CH2H4folate. Biochemistry In press 25. Lockshin, A., and Danenberg, P. V. (1980) Hydrodynamic behavior of human and bacterial thymidylate synthetases and thymidylate synthetase-5-fluoro-2’-deoxyuridylate-5,10-methylenetetrahydrofolate complexes. Evidence for large conformational changes during catalysis. Biochemistry 19, 4244-4251 26. Carreras, C. W., Climie, S. C., and Santi, D. V. (1992) Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation. Biochemistry 31, 6038-6044 27. Tatum, C., Vederas, J., Schleicher, E., Benkovie, S. J.,and Floss, H. (1977) Stereospecificity of thymidylate synthetase. .J. C. S. Chem. Commun. 218-220
35.
36.
37.
38.
39.
40.
41.
H. H., and Aull,J.
L. (1978)A
kinetic
study
of thymidy-
late synthase from Lactobacillus casei. j Biol. Chem. 253, 940-945 Beaudette, N. V., Langerman, N., and Kisliuk, R. L. (1980) A calorimetric study of the binding of 2-deoxyuridine-5-phosphate and its analogs to thymidylate synthetase. Arch. Biochem. Biophys. 200, 410-417 Kraulis, P. (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. j App!. Crystallogr. 24, 946-950 Ferone, R., Hanlon, M. H., and Singer, S. C. (1986) a-Carboxyllinked glutamates in the folylpolyglutamates of Escherichia coil thymidylate synthase. J. BioL Chem. 261, 16356-16362 Michaels, M. L., Kim, C. W., Matthews, D. A., and Miler,J. H. (1990) Escherichia coii thymidylate synthase: amino acid substitutions by suppression of amber nonsence mutations. Proc. Nat!. AcaiL Sd. USA 87, 3957-3961 Kim, C. W., Miehaels, M. L., and Miller, J. H. (1992) Amino acid substitution analysis of E. coil thymidylate synthase: the study of a highly conserved region at the N-terminus. Proteins: Struct. Funct. Genet. 13, 352-363 Dev, I. K., Yates, B. B., Leong, J.,and Dallas, W. S. (1988) Functional role of cysteine 146 in Escherichia coil thymidylate synthase. Proc. NatL Acad. Sc USA 85, 1472-1476 LaPat-Polasko, L., Maley, G. F., and Maley, F. (1990) Properties of bacteriophage T4 thymidylate synthase following mutagenic changes in the active site and folate binding region. Biochemistry 29, 9561-9572 Sand, D. V., Pinter, K., Kealey, J., and Davisson, V. J. (1990) Site-directed mutagenesis of arginine 179 of thymidylate synthase. j Biol. Chem. 265, 6770-6775 Zhang, H., Cisneros, R.J., Deng, W.,Johnson, L. F., and Dunlap, R. B. (1990) Site-directed mutagenesis of mouse thymidylate synthase: alteration of Arg 44 to Val 44 in a conserved loop guarding the active site has striking effects on catalysis and nueleotide binding. Biochem. Biophys. Res. Commun. 167, 869-875 Liu, L., and Santi, D. V. (1992) Mutation of asparagine 229 to aspartate in thymidylate synthase converts the enzyme to a deoxycytidylate methylase. Biochemistry 31, 5100-5104 Hardy, L. W., and Nalivaika, E. (1992) Asn177 in Escherichia coli thymidylate synthase is a major determinant of pyrimidine specificity. Proc. Nat!. Acad Sci. USA 89, 9725-9729 Climie, S. C., Carreras, C. W., and Santi, D. V. (1992) Complete replacement set of amino acids at the C-terminus of thymidylate synthase: quantitative structure-activity relationship of mutants of an enzyme. Biochemistry 31, 6032-6038 Dev, I. K., Yates, B. B., Atashi, J., and Dallas, W. 5. (1989) Catalytic role of histidine 147 in Escherichia coil thymidylate synthase. j Biol. Che,n. 264, 19132-19137
677
