Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase

Shido Kawase, Sung-Woo Cho1, James Rozelle, Robert M. Stroud, Janet Finer-Moore and Daniel V. Santi2

Department of Biochemistry and Biophysics and Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143-0448, USA and 1 Department of Biochemistry, College of Medicine, University of Ulsan, Seoul 138-040, Korea


    Abstract
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 Abstract
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 Materials and methods
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Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2.1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP. In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS. Mutations of R23 increase Km for dUMP 2–20-fold, increase Km for cofactor 8–40-fold and decrease kcat 9–20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme. Mutations of R178 increase Km for dUMP 40–2000-fold, increase Km for cofactor 3–20-fold and do not significantly affect kcat. These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues. Kinetic parameters for all R179 mutations except R179P were not significantly different from wild-type values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues. R218 is essential for the structure of the catalytic site and all mutations of this arginine except R218K were inactive.

Keywords: dUMP/electrostatic/hydrogen bond/saturation mutagenesis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the dimeric enzyme thymidylate synthase (TS), two conserved arginines from each protomer (in Lactobacillus casei TS, R23, R218 at the active site and R178 and R179 from the other protomer) are hydrogen bond donors to the phosphate moiety of dUMP (Figure 1Go). However, each of these arginine residues plays a distinctive structural or functional role, as evidenced by the different effects of mutating each site (Climie et al., 1990Go; Michaels et al., 1990Go; Santi et al., 1990Go). Several studies have appeared on aspects of the structure and function of these conserved arginines (Santi et al., 1990Go; Zhang et al., 1990Go; Lemeignan et al., 1993Go; Finer-Moore et al., 1996Go; Strop et al., 1997Go; Morse et al., 2000Go). These studies suggest that at least three of the phosphate-binding arginines, R23, R178 and R179, contribute to catalysis primarily through binding and orienting substrate, 2'-deoxyuridine 5'-monophosphate (dUMP), and cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate). Since R23, R178 and R179 are distant from the active site cysteine and from the site of methylation, they would not be expected to have a direct role in the chemistry of the enzyme reaction.



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Fig. 1. Schematic diagram showing hydrogen-bonding interactions (dashed lines) between dUMP and TS.

 
Mutant replacement sets at these arginines have been surveyed for activity using complementation assays (Climie et al., 1990Go; Michaels et al., 1990Go). However, detailed kinetic studies have involved a few (four at most) variants at each site, hence they give an incomplete picture of the contributions of the arginines to the reaction. Studies of a limited number of substitutions at a particular residue can give misleading results. We have therefore embarked on a comprehensive study of the structure–function relationships of the phosphate-binding arginines with emphasis on substrate binding, nucleophilic attack of the catalytic cysteine on dUMP and the final step of the reaction that is believed to reflect kcat. These steps in catalysis are expected to be particularly dependent on the contributions of affinity and substrate orientation provided by the arginines.

As with other TS residues studied in our laboratories, the approach is to prepare and characterize a `replacement set' of different mutants at the target residues. We use biochemical assays to dissect contributions of the mutated residues to individual steps in the reaction. Interpretations of the effects of mutations are made with reference to crystal structures of TS complexes that are structural analogs of intermediates in the reaction. In this paper, we describe the preparation and steady-state characterization of replacement sets of R23, 178, 179 and 218 mutants. We provide atomic-level interpretations of the effects of mutations on binding of substrates and catalysis that are consistent with all information on the enzyme reported to date.


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Materials

Oligonucleotides were synthesized and purified at the UCSF Biomolecular Resource Center. Phosphocellulose (P11) was purchased from Whatman. (6R)-CH2H4folate was a gift from Eprova AG Research Institute. The Escherichia coli strain DH5{alpha} was obtained from Gibco-BRL. The TS-deficient E.coli strain {chi}2913recA ({Delta}thyA572, recA56) has been reported (Climie and Santi, 1990Go). Plasmids pSCTS9 containing a synthetic L.casei TS gene have been reported (Climie and Santi, 1990Go; Climie et al., 1992Go). All other materials were the purest available from commercial sources.

DNA manipulations

General methods for DNA manipulations and bacterial transformation were as described (Sambrook et al., 1989Go). Automated DNA sequencing was performed at the UCSF Biomolecular Resource Center.

Mutagenesis

Cassette mutagenesis was performed as described previously (Climie et al., 1990Go). Generally, a fragment of the synthetic TS gene containing the target residue was excised and replaced by a synthetic oligonucleotide duplex that contained NN(G + C) at the target codon. The ligation mixture was used to transform E.coli DH5{alpha}, plasmid pools were isolated and used to transform the TS-deficient strain {chi}2913recA. For the R23, 178 and 218 mutants, it was possible to apply restriction purification to ligation mixtures and/or plasmid pools at unique sites of the parent plasmid to optimize mutagenesis efficiency. Active mutants could be identified by the ability of clones to grow in minimal medium not containing thymine.

To facilitate the construction of R23 mutants, pSCTS9 was converted to pSCTS13st. First an XbaI site was introduced at codon 14 and then the XbaI/AflII fragment was replaced with a synthetic duplex which contained a silent NaeI site at the codons 32/33 and an internal NotI site to be used for restriction purification. A synthetic double-stranded oligonucleotide cassette which restored the coding sequence for TS and contained NN(G + C) at codon 23 was ligated into the XbaI–NaeI digested vector. The ligation mixture was digested with NotI and transformed into E.coli DH5{alpha}. Plasmid DNA was prepared from pooled transformants, digested again with NotI and transformed into {chi}2913recA. Plasmid DNA was prepared from isolated clones and R23 mutants were identified by DNA sequence analysis.

R178 mutants were prepared from a derivative of pSCTS9, pSCTS9 (SmaI), which contains a SmaI site at nucleotide 523 and consequent Y176G mutation. A synthetic oligonucleotide duplex which restored Y176 and contained NN(G + C) at codon 178 was ligated into the SmaI–Bsu36I digested vector. A unique SalI site spans the R178 codon of the parent pSCTS9, so the ligated mixture was digested with SalI to destroy any remaining parent vector and the mixture was used to transform E.coli DH5{alpha} cells. Transformants were pooled and plasmid DNA was isolated, re-digested with SalI and used to transform E.coli {chi}2913recA. Plasmid DNA was prepared from isolated clones and R178 mutants were identified by DNA sequence analysis.

R179 mutants were constructed by replacing the SmaI–Bsu36I fragment of pSCTS9/SmaI with a synthetic oligonucleotide duplex which contained NN(G + C) at codon 179 (Climie et al., 1990Go). The ligation mixture was used to transform E.coli DH5{alpha} cells, transformants were combined and the plasmid pool was used to transform E.coli {chi}2913recA. Plasmid DNA was prepared from isolated clones and R179 mutants were identified by DNA sequencing.

R218 mutants were obtained by replacing the AvrII–PstI fragment of pSCTS9 with a synthetic oligonucleotide duplex which contained NN(G + C) at codon 218. A unique BglII site spans the R1218 codon of the parent pSCTS9, so the ligated mixture was digested with BglII to destroy any remaining parent vector. The ligation mixture was used to transform E.coli DH5{alpha} cells, transformants were combined and the plasmid pool was used to transform E.coli {chi}2913recA. Plasmid DNA was prepared from isolated clones and R218 mutants were identified by DNA sequencing.

Protein purification

Enzymes were expressed in E.coli {chi}2913recA and purified using automated sequential chromatography on phosphocellulose and hydroxyapatite (Kealey and Santi, 1992Go). The purification was monitored by SDS–12.5% PAGE. Enzymes were concentrated with Amicon Centriprep-30 concentrators and stored at –80°C until use. Generally, we obtained 10–50 mg of protein per liter of culture that was >95% homogeneous by SDS–PAGE. Enzyme concentration was measured spectrophotometrically using the extinction coefficient for wild-type TS of {varepsilon}278 = 125 600 l/mol.cm (Carreras et al., 1994Go). It was assumed that mutations of arginine residues had no significant effect on this value.

Complementation of mutants

Individual colonies were streaked on duplicate minimal agar plates containing 50 µg/ml ampicillin with and without 50 µg/ml thymine. Transformants producing active TS were identified by the ability of Thy E.coli {chi}2913recA cells to grow on minimal agar plates lacking thymine.

dTMP formation

Thymidine-5'-monophosphate (dTMP) formation was assayed spectrophotometrically at 25°C (Pogolotti et al., 1986Go). One unit of TS activity catalyzes the formation of 1 µmol of dTMP per minute in 1 ml of reaction mixture. Unless specified otherwise, CH2H4folate was kept constant at 300 µM when the concentration of dUMP was varied and the concentration of dUMP was fixed at 100 µM when CH2H4folate was varied. The kcat and Km values for cofactor were obtained from experiments varying CH2H4folate in the presence of a large excess of dUMP (>20Km). As indicated, saturating concentrations of CH2H4folate were sometimes not feasible when varying dUMP because of the high Km values exhibited by some mutants. In such cases, kcat values were calculated by correcting for the non-saturating CH2H4folate using the equation for a sequential ordered mechanism, Vmax = Vapp/(1 + KmB/[B]) (Segel, 1975Go).

Dehalogenation of BrdUMP

TS-catalyzed dehalogenation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP) (Garret et al., 1979Go) was performed at 25°C in a buffer containing 50 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) (pH 7.4), 25 mM MgCl2, 1 mM EDTA, 6.5 mM formaldehyde and 10 mM dithiothreitol (DTT). Dehalogenation was verified by the spectrum shift caused by conversion of BrdUMP to dUMP. Kinetic assays were performed by monitoring the decrease in absorbance at 285 nm, using {varepsilon}285 = 5320 l/mol.cm (Garret et al., 1979Go). One unit of TS activity catalyzes the dehalogenation of 1 µmol of BrdUMP per minute in 1 ml of reaction mixture. For the determination of kinetic parameters, the concentration of TS was 5–10 µM and the concentration of BrdUMP was varied up to 160 µM.

PLP binding

Dissociation constants for dUMP were determined by displacement of pyridoxal 5'-phosphate (PLP) from the TS–PLP complex as described (Liu and Santi, 1993Go; Santi et al., 1993Go).

Apparatus

All spectrophotometric measurements were made on a Hewlett-Packard Model 8452A diode-array spectrophotometer. Steady-state kinetic parameters and dissociation constants were determined from a non-linear least-squares fit of the data to the appropriate equation for each reaction using the program Kaleidagraph (Abelbeck Software) on a Macintosh computer.


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Mutagenesis and genetic complementation

Mutants were constructed by cassette mutagenesis of synthetic TS genes using oligonucleotides that contained a mixture of 32 codons, NN[C + G], in place of the target codons. NN[C + G] encodes all 20 amino acids and the amber stop codon (Climie et al., 1990Go). After transformation of the TS-deficient strain E.coli {chi}2913recA, colonies were randomly subjected to DNA sequencing. We obtained 15 mutants each of R23 and R218 and 16 each of R178 and R179, which were deemed sufficient for the present study.

The mutants were examined by genetic complementation of TS deficient E.coli {chi}2913recA on minimal agar in the absence of exogenous thymidine. Only three of the 15 R23 mutants (R23I, K and Q) expressed sufficient TS activity to support growth. However, the growth of {chi}2913recA transformed by these mutants was extremely slow, indicating that the in vivo activities were barely sufficient to support growth. Of 16 R178 mutants studied, only two (R178K and T) supported the growth of TS-deficient cells. Only one (R179P) of the 16 R179 mutants failed to support growth and none of the 15 R218 mutants supported the growth of TS-deficient cells.

Protein purification

E.coli {chi}2913recA transformed by individual arginine mutant plasmids were grown in 1 l of LB medium for 24 h. SDS–PAGE analysis of extracts showed that all of the mutant enzymes were expressed at levels of 10–50% of the total soluble protein. Each mutant TS was purified using sequential phosphocellulose–hydroxyapatite chromatography (Kealey and Santi, 1992Go) and was more than 95% homogeneous on SDS–PAGE.

dTMP formation

Steady-state kinetic parameters for dTMP formation by the purified arginine mutants and wild-type TS were determined (Table IGo). The steady-state kinetic parameters for R23I, R178T and R179T have been reported previously (Morse et al., 2000Go), but are included in Table IGo for convenience. The kcat values of the R23 mutants were reduced 9–120-fold compared with wild-type, while the Km values were 2–20-fold higher for dUMP and 8–40-fold higher for CH2H4folate. Compared with wild-type TS, the kcat/Km values were decreased 70–1800-fold for dUMP and 10–2800-fold for CH2H4folate.


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Table I. Kinetic and binding constants for L.casei TS R23 and R178 mutants
 
Steady-state kinetic parameters for dTMP formation by 10 selected R178 mutants showed that five (R178D, E, G, L and W) were below the lower limit of detection by the spectrophotometric assay (<0.003 s–1). The others exhibited 40–2000-fold increased Km values for dUMP compared with wild-type TS, but less than a 3-fold reduction in kcat. The Km values for CH2H4folate were increased 3–20-fold and kcat/ Km was reduced 30–4000-fold for dUMP and 2–40-fold for CH2H4folate.

With the exception of R179P, all of the 16 R179 mutants exhibited kinetic parameters that differed from the wild-type values by less than 10-fold. R179P showed a kcat ~3000-fold lower than wild-type TS. R179A and R179T had kcat values nearly identical with wild-type TS and, except for R179P, the lowest kcat value (R179F) was only 3.7-fold lower than the wild-type enzyme. R179E had the highest Km value for dUMP, but this was only 8-fold greater than wild-type TS. The Km values for CH2H4folate differed from wild-type enzyme by less than 3-fold. With the exception of R179P, the kcat/Km values were 2.8–15-fold lower for dUMP; R179A had a slightly higher kcat/Km for CH2H4folate, but other mutants were 1.5–9-fold lower than wild-type TS.

The apparent kcat values of the 10 R218 mutants studied were reduced 30- (R218K) to >10 000-fold compared with wild-type TS. With the exception of R218K, which had a 100-fold increase in Km of dUMP and similar Km for CH2H4folate, the very low activities of the mutants did not allow the determination of Km values.

Kd of dUMP

We attempted to measure Kd values for dUMP for the TS arginine mutants by competitive displacement of PLP from preformed enzyme–PLP complexes (Table IGo). All R23 mutants examined bound PLP and the Kd values for enzyme–dUMP complexes were decreased 30–100-fold compared with wild-type TS. With the R178 mutants examined, only R178K bound to PLP and with the R179 substitutions, only R179K, A and W bound PLP. None of the R218 mutants bound PLP.

Dehalogenation of BrdUMP

Kinetic parameters for the cofactor-independent debromination of BrdUMP were not significantly impaired in the R23 mutants. Most Km values were similar to wild-type, with the highest increased only 5-fold; kcat values were decreased <2-fold of the wild-type TS. None of the R178 mutants catalyzed the dehalogenation of BrdUMP. With the exception of R179P, all R179 mutants catalyzed debromination. However, kcat values were decreased 3 to >27-fold compared with wild-type TS and Km values were 2–10-fold higher. The negatively charged mutations, R179D and E, were very poor catalysts for debromination. None of the nine R218 mutants tested catalyzed the dehalogenation.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Using a synthetic L.casei TS gene and cassette mutagenesis with appropriate degenerate NN[C + G] codons, we prepared replacement sets at residues 23, 178, 179 and 218 of L.casei TS. For each of the positions modified, we selected a number of mutants that represented diverse side chains, purified each to homogeneity and determined steady-state kinetic and binding parameters. The spectrum of activities shown by the mutants at a given arginine site suggests the unique role that the arginine may have in catalysis. For example, mutations of R23 have little impact on dUMP binding, but have moderate effects on cofactor binding and on catalytic rate, indicating that this arginine is most important for later steps in the reaction. In contrast, those R178 mutants whose kinetics we measured had greatly elevated Kms for dUMP but kcats close to wild-type values, suggesting that R178 contributes mainly to dUMP binding.

We interpret our data in the context of apo-, dUMP-bound and ternary complexes of wild-type TS. These structures (which are analogs of reaction intermediates) show that the TS active site progressively closes as it sequentially binds dUMP and CH2H4folate. Since the conformations and packing of some of the arginine side chains are different in the three different conformations of the enzyme, the whole series of structures is required for understanding the arginines' role in catalysis at a molecular level.

Arg23

R23 is the phosphate-binding residue most affected by the conformational changes that accompany ligand binding. R23 is contained within a flexible loop consisting of residues 21–26. In apo-TS, the loop exhibits high thermal motion and the side chain of R23 is completely disordered. The loop, including the R23 side chain, becomes progressively better ordered as substrate, then cofactor bind. In L.casei TS, R23 is drawn into the binding site as dUMP binds, where it hydrogen bonds to the phosphate moiety in either of two distinct conformations. Upon binding of cofactor, a conformational change shifts the R23 loop and several other segments of the protein toward the active site and sequesters the ligands from bulk solvent. The C-terminus of the enzyme moves 5 Å towards the active site, where it forms hydrogen bonds to the cofactor and to the R23 guanidinium group. In TS ternary complexes with substrate and cofactor, R23 adopts a single, well-ordered conformation in which it forms salt bridges or hydrogen bonds with the phosphate of dUMP, the C-terminus and the pterin ring of the cofactor.

Since the lowest Km of any of the mutants is only ~2-fold higher (R23Y) than wild-type TS, R23 per se does not contribute much to binding of nucleotide. The Km values in the cofactor-independent dehalogenation of BrdUMP, which reflect Kd values for the binary enzyme–BrdUMP complex, are also not much different in the R23 mutants than in the wild-type enzyme. The same is true for the kcat of BrdUMP dehalogenation, indicating that the mutations do not significantly affect orientation of the substrate in the binary complex (Finer-Moore et al., 1998Go). As previously proposed for R179 mutants, upon loss of R23 hydrogen bonds to the phosphate of dUMP or BrdUMP, water or other hydrogen bond donors may provide compensating interactions with dUMP. In addition, the loss of electrostatic binding energy when R23 is mutated may be offset by the simultaneous reduction in unfavorable electrostatic repulsion among the phosphate-binding arginines (Morse et al., 2000Go). All of the R23 mutants examined bound PLP and showed the UV spectrum characteristic of hemithioacetal formation. Hence R23 is not essential for either dUMP or PLP binding.

The deleterious effects of R23 on cofactor binding could be due to one or several factors. One is that in the enzyme–nucleotide complex, dUMP may be bound to the mutants in a perturbed orientation; since dUMP provides a contact surface for cofactor binding, perturbation of its orientation is often accompanied by a decrease in cofactor affinity (Agarwalla et al., 1997Go). However, the similarity of the kcat values of the mutants and wild-type enzymes in BrdUMP dehalogenation argue against the importance of this factor. Second, loss of the interaction between R23 and the cofactor could directly contribute to lower affinity. Lastly, since R23 hydrogen bonds to the C-terminus to stabilize the ternary complex, the decreased cofactor binding may be secondary to a less efficient C-terminal closure. Mutants of V316 also destabilize the closed form and likewise show decreased cofactor binding (Climie et al., 1992Go).

Since the highest kcat of any of the mutants is ~10-fold lower than wild-type TS, the maximum contribution of the guanidinium group to kcat is <1 kcal/mol. Recently, the rate-determining step for dTMP formation has been shown to occur during hydride transfer from 5,6,7,8-tetrahydrofolate (H4folate) to the exocyclic methylene intermediate (Spencer et al., 1997Go). Thus, the change in kcat must result from either a change in the concentration of an intermediate formed prior to hydride transfer or a change in the free energy of activation at the rate-determining hydride transfer step. The lower kcat values of R23 mutants could simply be due to a lower steady-state concentration of the closed form of the enzyme resulting from the absence of R23 stabilization. More likely, they may be due to an aberrant conformation of the cofactor in the ternary covalent complex that is sub-optimal for hydride transfer. The aberrant conformation could result from the loss of the water-mediated hydrogen bond from R23 to the cofactor or from increased mobility of the cofactor in the closed form of the enzyme (see above). Evidence that a step involving cofactor binding and/or C-terminal closure is responsible for the lowered kcat values is the observation that the kcat values for BrdUMP dehalogenation (which is cofactor and C-terminal closure independent) are not affected by mutations at R23. Taken together, the high cofactor Km values and low kcat values result in catalytic efficiencies (kcat/Km) that are 100–3000-fold lower than wild-type. With a few exceptions, these are insufficient to support cell growth and provide an explanation of why R23 of TS has been highly conserved.

Arg 178

In addition to binding the phosphate of dUMP, R178 interfaces with several other residues of TS that are important in nucleotide binding. In the binary complex with dUMP, R178 is also in van der Waals or hydrogen bonding contact with Y261 and H259, which are hydrogen bonded to the 3'OH of dUMP, S219, which is hydrogen bonded to phosphate and D22, which may contribute to positioning the R23 loop (residues 21–26) in the closed ternary complex. As the R23 loop moves into the active site when cofactor binds, R178 becomes more completely buried by protein or ordered water molecules and is in closer contact with the above-mentioned residues. In ternary complexes of E.coli TS with dUMP and cofactor analogs, R178 is completely inaccessible to bulk solvent and is hydrogen bonded to both Y261 and D22.

Other than lysine, the only residues tolerated at residue 178 have small neutral side chains. In the mutants studied, the Km values for dUMP were very high. Likewise, none of the mutants catalyzed the cofactor independent dehalogenation of BrdUMP. It has been suggested that the absence of dehalogenation correlates with a perturbed orientation of the nucleotide in the binary complex (Finer-Moore et al., 1998Go). The very large effects on nucleotide binding probably reflect the direct interactions of R178 with phosphate as well as structure-stabilizing interactions with other groups that are involved in dUMP binding, i.e. Y261, S219, H259 and the loop comprised of residues D22 through T26.

The Km values for cofactor in the R178 mutants were also moderately increased, but not nearly to the extent observed for dUMP. Although R178 has no direct contact with the cofactor, its prominent structural role in ternary complex conformations of TS may account for effects of R178 mutation on Km for CH2H4folate. In addition, since dUMP provides a contact face for the cofactor, aberrant orientation of the nucleotide in the binary complexes of the R178 mutants could lead to the decreased affinity for the cofactor.

In view of the effects of R178 mutations on binding of substrates, it is surprising that they have so little effect (<3-fold) on kcat. We suggest that binding of cofactor and closing of the C-terminus corrects any unfavorable orientations of the nucleotides in binary complexes of the R178 mutants. In this circumstance, regardless of the degree of dUMP misorientation in binary complexes, the ternary complex would possess a favorable orientation for catalysis. There is, in fact, evidence that R178 mutants that cannot bind dUMP alone in an orientation favorable for catalysis, can be induced by cofactor binding to form productive ternary complexes (Strop et al., 1997Go). In a crystal structure of the essentially inactive E.coli TS mutant R178E, the E178 side chain occludes the phosphate-binding site of the enzyme, explaining why binary complexes with dUMP could not be crystallized. However, when a cofactor analog was included in the crystallization, both the cofactor analog and dUMP were present in the crystal structure. Furthermore, both the cofactor analog and the pyrimidine ring of the dUMP were bound in their wild-type binding sites with the catalytic cysteine covalently bonded to dUMP. In this case, ternary complex formation was deemed rate determining and responsible for the low enzyme activity (Strop et al., 1997Go).

Arg 179

Of the four arginine residues that are hydrogen bonded to the phosphate of dUMP, R179 is the only one that appears to have the sole function of binding dUMP. The side chain of R179 is exposed to bulk solvent on one side and is not in van der Waals or hydrogen-bonding contact with other ligand-binding residues (besides the other phosphate-binding arginines) in the free enzyme or in binary or ternary complexes. The structural environment of R179 is not significantly changed by the conformation change that occurs when the ternary complex forms: it stabilizes binary and ternary complex forms of the enzyme equally and would not be expected to affect the energetics of cofactor binding.

Mutant replacement sets of R179 in E.coli and L.casei TS's indicate that many substitutions permit growth of TS-deficient cells, but these mutants were not studied in detail (Climie et al., 1990Go; Michaels et al., 1990Go). The results of the present work are in agreement with a study on a smaller number of mutants that concluded that there is little perturbation of the structure of the dUMP binding site upon modification of R179 (Finer-Moore et al., 1996Go). For R179P, where Km for dUMP is increased 40-fold and where the dehalogenation of BrdUMP is not observed, there may be a significant change in the secondary structure of the protein.

One unusual finding is that of the R179 mutants, only Ala, Lys and Trp bind and react with PLP. This result reflects a thus far unique, differential contribution of a TS residue to PLP and dUMP binding. The greater contribution of R179 to PLP binding could be explained if, in the TS–PLP complex, as in the complex of TS with inorganic phosphate, only R218, R178 and R179 bind to the phosphate moiety of the ligand (the dUMP phosphate moiety also binds to R23 in wild-type TS–dUMP complexes). In this case, mutation of R179 would leave only two arginines to bind to PLP (R218 and R178) but three to bind to dUMP (R218, R178 and R23). We have recently shown a very large energetic difference in dUMP binding between two and three arginines at the phosphate-binding site (Morse et al., 2000Go). Thus mutation of R179 could lead to moderate decrease in the binding affinity for dUMP, but a very large decrease in the binding affinity for PLP.

Another unusual feature of the R179 mutants is the very low kcat values observed in the dehalogenation of BrdUMP, especially pronounced with the negative residues Asp and Glu. Lower kcat values for dehalogenation are usually characteristic of mutants that bind dUMP in an aberrant conformation. Greater vibrational entropy of the nucleotide when bound to some of the mutants (e.g. R179E), as indicated by high B-factors for dUMP in structures of their binary complexes (Finer-Moore et al., 1996Go), may contribute to the reduction in kcat for dehalogenation, but entropic effects are not seen in the other kinetic parameters. Furthermore, in the structures of some of the other R179 mutants, dUMP atoms have B-factors comparable to wild-type values (Finer-Moore et al., 1996Go; Morse et al., 2000Go). The unusual effects of R179 in PLP binding and the dehalogenation reaction have not been revealed in previous studies and are distinct from effects seen with mutations at the other arginine residues.

Structural studies have not revealed direct or indirect interactions between R179 and the cofactor or other protein residues that might affect binding of the cofactor (Matthews et al., 1990Go; Montfort et al., 1990Go). In accord with such studies, the R179 mutants show cofactor Km values within ~3-fold of wild-type TS. With the exception of R179P, all R179 mutants show kcat values within 3-fold of wild-type TS. Thus, once the ternary complex has formed, it is competent for catalysis.

Arg 218

R218 is a buried residue at the dimer interface of the enzyme, in close proximity to the active site C198. The R218 guanidinium group forms hydrogen bonds with two of the phosphate oxygens in dUMP, with the backbone carbonyl oxygens of P197 and P196 (Ala in E.coli TS) and across the dimer interface to the backbone carbonyl of R178 (Hardy et al., 1987Go; Finer-Moore et al., 1993). Thus, in addition to direct interactions with the phosphate, R218 helps stabilize the conformations of two critical loops in the enzyme: the segment containing the catalytic cysteine, C198 and the segment containing two of the other three phosphate-binding arginines. There are no direct interactions between R218 and the cofactor.

The hydrogen bonds formed between R218 and adjacent segments of the peptide backbone suggest that, in addition to direct phosphate interactions, the residue serves an important role in maintaining the structure of the active site cavity. Mutation of R218 should result in disorder or rearrangement of residues in the active site that contribute to nucleotide and PLP binding. Indeed, preliminary crystallographic results indicate significant structural perturbation of the active site cavity of R218K (D.L.Birdsall, personal communication). Thus, in addition to direct contributions to phosphate binding, R218 seems to coordinate the active site in the structure necessary to bind nucleotide. This important structural role of R218 may be why all mutations at this residue decrease kcat/Km by at least 3000-fold. We have also postulated that R218 increases the nucleophilicity of C198 to facilitate addition to C6 of the pyrimidine ring (Hardy et al., 1987Go) and we cannot rule out this role in catalysis.

Conclusion

The four phosphate-binding arginines of TS provide a favorable electrostatic environment for the phosphate moiety of the substrate, dUMP. Because the arginine guanidinium group is capable of forming multiple hydrogen bonds, the phosphate-binding arginines are intricately connected not only to dUMP, but also to surrounding protein residues that are important for cofactor binding and catalysis. Thus mutations of the arginines not only affect dUMP binding and orientation, but also indirectly affect Km for cofactor and kcat. The effects of mutations of these arginines on kinetic constants of TS are different for each arginine and we have rationalized them by considering the structural role of each arginine in binary and ternary complexes of the enzyme.

Mutations of the two most buried residues, R178 and R218, which hydrogen bond to many other residues in the active site cavity of the enzyme, have the largest impact on Kms. They are important for maintaining the optimum shape and distribution of binding determinants in the active site cavity. R23 has a direct role in cofactor binding and orientation by virtue of its role in a hydrogen-bond network coordinating the cofactor pterin ring and the C-terminus of the enzyme. The importance of this network is reflected in moderate increases in Km for cofactor and ~10–120-fold decreases in kcat for the R23 mutants. The external residue R179 forms hydrogen bonds only to the phosphate moiety of dUMP, hence mutations of this residue affect dUMP binding affinity, but have little impact on Km for cofactor or kcat.


    Notes
 
2 To whom correspondence should be addressed Back


    Acknowledgments
 
This work was supported by Public Health Service Grants CA-14394 (to D.V.S.) and CA-41323 (to J.F.-M. and R.M.S.) from the National Institutes of Health. S.K. was supported by UNITIKA Ltd, Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received February 4, 2000; revised May 12, 2000; accepted May 15, 2000.