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Mol. Cells, Vol. 2, pp. 109-114 Site-directed Mutagenesis of Arginine-178 of Thymidylate Synthase from Lactobacillus casei Sung-Woo Cho*, and Soo Young ChoP Department of Biochemistry, College of Medicine, University of Ulsan. Seoul 138-040, Korea; IDepartment of Genetic Engineering. College of Natural Science, Hallym University, Chunchon 200-702, Korea (Received on February 23, 1992) X-ray structural studies have previously shown that Arg-178 of thymidylate synthase interacts with bound inorganic phosphate or with the S'-phosphate of the bound substrate dUMP. The importance of Arg-178 to the structure/function of thymidylate synthase is also indicated by its complete conservation among the 17 thymidylate synthases thus far sequenced. In the present work, cassette mutagenesis has been performed for Arg-178 of Lactobacillus casei thymidylate synthase. Eleven amino acid substitutions' have been obtained for Arg-178. Methods have been developed for determination of functional and phenotypical characteristics for each of the newly synthesized mutant proteins. Functionally acceptable substitutions were defined by genetic complementation of thymidylate synthase deficient cells and further characterized by determination of specific activity. Evaluation of kinetic parameters of the mutants was performed in crude extract using S-fluoro-2'-deoxyuridylate as an active site titrant Analysis of the mutants by genetic complementation indicates that thymidylate synthase can tolerate a number of amino acid substitutions at that position and shows that Arg-178 is not strictly required for catalytic activity. is supported by the fact that this residue is completely Thymidylate synthase (EC 2.1.1.4S) catalyzes conversion of dUMP and S,lO-methylenetetrahydrofolate conserved in all TS' sequenced to date (Bzik et al., 1987). (CH 2H.Jolate) to dTMP and 7,8-dihydrofolate (H2folate). The catalytic mechanism of thymidylate synthase · In this report, we describe the strategy and preliminary results of an approach to understanding the st(IS) has been extensively studied (Santi and Danenructure of TS by saturation site-directed mutagenesis. berg, 1984; Santi et al., 1987). The amino acid sequen"Replacement sets" were constructed in which Arg-178 ces of the enzyme from some 17 sources are known, and the three-dimensional structures of TS from severesidue was replaced by a large number of substitural sources have been solved (Hardy et al., 1987). The tions. The mutagenic DNA cassette contained a mixprimary sequences of TS have revealed that it is one ture of 32 codons that encode 20 amino acids and the amber (TAG) stop codon at the target site (Reiof the most conserved proteins known; the similarity dhaar-Olson and Sauer, 1988). An advantage of synamong TS sequences implies a functional importance for the conserved residues. thetic TS gene as a mutagenesis vehicle (Climie and Santi, 1990) is demonstrated by the construction of The X-ray structures of the Lactobacillus casei TS-Pi complex and the Escherichia coli TS' dUMp· lO-propaa series of 11 site-directed mutations at Arg-178 in rgyl-S,8-dideazafolate ternary complex have provided a single experiment The use of synthetic gene is furinsight about the possible roles of specific residues ther enhanced by its high level ·of expression and by of the protein, especially 4 conserved Arg residues the ability to screen TS mutants by genetic comple(Arg-23, -178, -179, and -218 in L casel) which are mentation in E. coli. located within bonding distance of the liganded phosphate. Chemical modification and BC NMR studies Materials and Methods have also suggested that one or more arginine residues are involved in binding of dUMP (Cipollo and DunMaterials lap, 1979; Cipollo et al., 1982; Belfort et al., 1980). Two Restriction endonucleases, polynucleotide kinase, T4 of these, Arg-178 and Arg-179, emanate from the other DNA ligase, T4 DNA polymerase, and E. coli DNA subunit of the homodimeric enzyme. Hence, it is reapolymerase I were purchased from New England Biosonable to predict that Arg-178 may playa ' role in labs. [a-35S]dATP (>600 Ci/mmole) were purchased binding the phosphate moiety of dUMP. The putative from Amersham Corp. 'Sequenase DNA sequencing importance of Arg-178 to the structure/function of TS * To whom correspondence should be addressed The abbreviations used are: FdUMP, 5-fluoro-2'-deoxyuridylate; TS, thymidylate synthase. © 1992 The Korean Society of Molecular Biology Mutagenesis of Arg-178 in Thymidylate Synthase 110 kit was from U.S. Biochemicals. Oligonucleotides were synthesized at the University of California, San Francisco, Biomolecular Resource Center. Other materials have been reported (Bruice and Santi, 1982; Pinter et al., 1988) or were commonly available. Bacterial strains and plasmids Strain DH5a [cj>80IacZLlM15; ara, ~(lac-proAB), rpsL hsdR ; T. O. Baldwin, Texas A&M, College Station] was used as the host strain for plasmid-mediated transformations for the initial isolation of mutants. The Thy- E. coli strain X2913 (&hyA572), a gift from Dr. Daniel V. Santi, University of California, San Francisco, USA, was used to test plasmids for TS activity by genetic complementation and for the production of recombinant TS. Strain JM101 [sup£, th4 ~(lac proAB) (F' , traD, proA +B+, lac[QZLlM15)] was used to propagate M13 clones used for DNA sequencing. A synthetic DNA (PSCTS9) was obtained from Dr. Daniel V. Santi, University of California, San Francisco, USA ' Plasmid construction Mutations were performed by replacing the fragment with a synthetic DNA duplex (PSCTS9) that presented mixed bases for the codons of interest. Since the fragmeIit between two flanking sites (Clal and Mst II) was too large (80 bp), pSCTS9/Smal, a derivative of pSCTS9, was constructed by oligonucleotide-directed mutagenesis of synthetic TS gene cloned into M13 mpl9 vector (Fig. 1). The new construct denominated as M13mpI9ffS/Smal is the same as the wild type, except that Tyr-176 (TAC) is replaced by Gly (GGC) to make a Smal site (CCCGGG) at His-174 (CAC), r ._. Hind In £.coA I _CI. TS RI y--- D • Ria Hili Hln PLATE (IPTG + Xgal) r"'aq.... (In..ft) Stu. Plaq.... (NoInNfl) ~ 7 dNTPs O ) Sja' (Oligo) O=~"' -~, I eamHI • a5011 BomH I ,SS-DNA ~(TOmplOIO) ---====-. sm, I SOli b. '"." ...p . , I Figure 1. Construction of pSCTS9/SmaI 9:: I elmHI • &5011 Mol. Cells Pro-175 (CCG), and Gly-176 (GGC). Finally this TS/Smal gene in Ml3mp19 vector was sub cloned into pUCl8 to construct pSCTS9/SmaI plasmid. The modified DNA (PSCTS9/Smal) contains one additional unique restriction site (Smal) between the two restriction sites (Cia I and MstII). Therefore, the flanking sites used for Arg-178 mutation were Smal and MstII with a length of 36 bp in pSCTS9/SmaI. General methods for DNA manipulation were carried out as previously described by Maniatis et al. (1982). DNA fragments used for cloning were separated by electrophoresis on 0.8% low melting point agarose gels, and the excised bands were used directly in the ligation reaction (Crouse et aI., 1983). Mutagenesis of Atg-178 A series of single amino acid substitutions of Arg178 were obtained by cassette mutagenesis of plasmid pSCTS9/Smal (See Fig. 2). Plasmid DNA was digested with Smal and MstII to remove a 36-bp fragment, and vector DNA was purified by electrophoresis using 1% low melting point agarose. The 36-bp Smal/MstII fragment was replaced with a 36-bp synthetic DNA duplex containing multiple substitutions on both DNA strands at positions encoding Arg-178. The substitutions included a equal mixture of all four bases at the first and second positions of the codon and an equal mixture of G and C at the third position. Mutagenic oligonucleotides were annealed in 10-111 reaction mixtures that co~tained 1, 5, 10, 50, and 100 pmoles of each unphosphotylated oligonucleotide. The annealed DNA fragments were ligated with 0.5 Ilg of gelpurified vector DNA as described above and one half of each ligation reaction was used to transform strain DH5a to ampicillin resistance. The resulting colonies were pooled by flooding the plates with 3 rnl of L broth and collecting the cell suspension with a sterile pipette. Plasmid DNA was prepared from the pooled cells and used to transform E. coli strain X2913 (thy - ), which was then plated on LB agar containing 100 Ilg/rnl of ampicillin and 50 Ilg/rnl of thymidine. Plasmid DNA was prepared from individual X2913 transformants and Arg-178 mutants were identified by dideoxynucleotide sequencing using plasmid DNA as a template. Arg-178 mutants were further characterized by their ability to grow on minimal agar in the absence of thymidine. Protein preparations and enzyme assay For small scale preparations, the transformed E. coli strain X2913 (thy - ) was grown by inoculation of 50 rnl of LB containing 100 Ilg/rnl ampicillin and 50 Ilg/rnl thymidine with 0.25 rnl of overnight cultures and incubation for 16-20 h at 37 °e, Cells were harvested by centrifugation washed with cold 150 mM NaCl, then resuspended in 5 rnl of 100 mM Tris-HCl (PH 7.4), and 1 mM EDTA, and disrupted by sonication on ice bath; cell debris was removed by centrifugation at 10,000 X g for 15 min. Protein concentration was determined by the method of Read and Northcote (1981) using bovine serum albumin as a standard. TS Sung-Woo Cho & Soo Young Choi Vol. 2 (1992) ABC 0 A BCD Restrict B&C 111 zation of filters required approximately 24 h. Counting efficiencies were determined using external standards. Filtration efficiencies were routinely between 88 and 94% and were constant within one same experiment. Results ~ t.W Transformation ., 32codons 20 amino acids Sequence; screen/selecl for active mutant in TS·deficient cells Figure 2. Strategy for saturation site-directed mutagenesis of synthetic TS gene and identification of catalytically. active mutants. A fragment of the TS gene between two restriction sites was removed and replaced by a mixture of oligonucleotides containing NNG/C at the Arg-178 codon. The resultant plasmids were sequenced and screened in E. coli thy - cell (X2913) for catalytic activity. activity was assayed spectrophotometrically at 25 °C using the conditions of Pogolotti et al. (1986). One unit of activity is the amount of TS that catalyzes the formation of 1 mmole of product per min. TS synthesis was examined by SDS/PAGE of crude cell extracts that were prepared by sonication of the overnight cultures grown in L broth containing 100 Ilg/rnl of ampicillin and 50 Ilg/rnl of thymidine. SDS-PAGE was performed by the method of Laemrnli (1970) and gels were stained in Coomassie Brilliant Blue R-250. Filter binding assay The formation of the enzyme-CH)FdUMP complex was performed in a standard assay mixture containing 50 mM TES (PH 7.4), 25 mM MgCh, 6.5 mM HCHO, 1 mM 2-mercaptoethanol, 0.l5 mM FAIL, (6- 3H) FdUMP, and 10 Ilg of cell extract. The reaction was initiated by addition of the cell extract in a total assay volume of 100 j..ll. Mter 45-min incubation, 4O-j..ll aliquots were assayed in duplicate for complex formation and 10 j..ll was removed for determination of CH) FdUMP concentration in the assay mixture. Nitrocellulose membranes were soaked before use in 75 mM potassium phosphate (PH 7.4). Filters which were not wetted within 2 min were discarded. The filter discs were placed on a filter manifold (Hoeffer-Scientific) and a gentle vacuum was applied to remove excess moisture. Without removing the vacuum, filters were washed with 2 rnl of the same buffer and then 40 j..ll aliquots of the reaction mixture were applied to the discs and allowed to permeate the membrane. Mter washing the filters with 6 rnl of the same buffer, the damped filters were placed in scintillation vials and dissolved in 10 ml of Aquasol. Optimal solubili- Arginine-178 was chosen as a target for mutagenesis because it is highly conserved and because it hasputative role in phosphate binding as revealed by previous solution and structural studies. The strategy used in the construction and characterization of the replacement set is outlined in Figure 2. Following ligation of oligonucleotide mixtures and initial transformation, colonies were pooled, and the mutagenized plasmid DNA pool was recovered and used to transform E. coli X2913 (thy - ) to ampicillin resistance. Individual mutants were identified by DNA sequencing, and the resulting plasmids were used to re-transform X2913. Plasmid DNA from the secondary X2913 transformants was again sequenced and tested for TS activity. Passage of the mutagenized DNA mixtures through several rounds of transformation ensured segregation and repair of the heteroduplex DNA molecules that were created during the construction of the mutants. It was not practical to identifY all possible mutants in a replacement set by this strategy, which would require sequencing of over 160 clones to obtain a 95% confidence level of obtaining a complete set. Generally, 20-30 isolates of a replacement set ,,:,ere seq~enced to give 10-15 different mutants of a glVen reSIdue. The results obtained with regard to complementation of X2913 for the different mutants of Arg-178 are shown in Table 1. Mter sequencing 30 clones, we have isolated 11 of the 20 possible amino acid substitutions, 1 amber codon, and three synonymous Arg codons. The wild-type Arg codon, CGT, was not found in the 30 plasmids sequenced, indicating the mutagenesis efficiency was >97% in this experiment. The 11 mutants arose at a frequency close to that expected on the basis of the codon distribution in the mutagenic DNA cassette (data not shown). Of the 11 different amino acid mutations obtained for Arg178, 3 mutants (Lys, Tyr and Thr) were able to complement the thyA deletion in X2913 on minimal agar in the absence of thymidine, indicating that catalytically active TS was being synthesized. However, 8 mutants (Leu, Pro, Val, De, Ser, Asp, Glu, and Phe) did not grow under the same conditions, suggesting that the TS synthesized in these cells was incapable of providing sufficient thymidylate to sustain growth. The observation that at least three different residues can be substituted at position 178 without complete loss of activity allows us to conclude · that Arg-178 is not strictly essential for TS function. Analysis of crude cell extracts by SDS-PAGE showed that all 11 mutant plasmids encoding an amino acid substitution at position 178 directed the synthesis of a 37-kDa protein that comigrated with TS (Fig. 3). SDS-PAGE and activity measurements of crude extracts prepared from each of the mutants expressed 112 Mutagenesis of Arg-I78 in Thymidylate Synthase Mol. Cells Table 1. Complementation screen and kinetic properties of Arg-I78 mutants K.n (~) Amino acid substitution Genetic complementation Specific activity k"", (sec ') dUMP Wild type Lys-l78 Thr-178 Tyr-178 Active Active Active Active 0.22 0.18 0.13 0.11 4.2 1.9 1.9 1.8 2.5 3 l.l 5.2 25 18 55 19 42 Leu-178 Ile-178 Pro-178 Phe-178 Val-I 78 Ser-178 Asp-I 78 Glu-178 Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive <0.001 <0.001 <0.001 0.002 0.003 0.007 <0.001 <0.001 nda nd nd nd 0.11 0.31 nd nd nd nd nd nd 22 1.5 150.9 nd nd nd nd nd nd 1253 996 nd nd CH 2H,Jolate k""t!K,,, dUMP (k ,) (~ - ' sec - ') CH 2 H,Jolate 1.68 0.06 0.23 0.07 0.23 0.03 0.11 0.04 nd nd nd nd 0.0005 0.0021 nd nd nd nd nd nd 0.0001 0.0003 nd nd and; not detectable. AmWT K Y T L P V 5 0 E F 97 kDa--" 66 kDa--" 45 kDa--" 31 kDa--" 22 kDa--" Figure 3. SDS-PAGE analysis of TS synthesis from X2913. Crude extracts from various Arg-I78 mutants; indicated by oneletter codes for the substituted amino acids except for the wild type (WT) and amber (Am). Lane I, molecular weight standard proteins; lane 9, purified TS. in X2913 showed that TS was expressed to a level of -10% of the total soluble protein except the amber substitution. Thus, the absence of catalytic activity is not due to a lack of production or stability of the different mutants. Results on catalytically active enzymes were further assessed by enzymatic assays of cell extracts. We have determined that minimal detectable specific activity in cell extracts is approximately I X 10- 4 U/mg protein, value 2,500 times lower than what has been observed for wild-type TS. Further characterization of functionally active TS presenting the different substitutions consisted in performing an estimation of k cat for the different mutant TS'. Determination of k ca, involves measurements of the actual amount of TS present in cell extracts. This was performed by measuring the formation of FdUMP-enzyme complex using the ftlter binding assay. We have routinely measured the formation of complex at 3 different FdUMP concentrations; 76.3, 381.5, and 763 nM. At 76.3 nM, FdUMP is below saturation and therefore provides values in order to calculate ftltration efficiency for each mutant. Calculations of the pmoles of dimer/mg cell protein were performed assuming that the stoichiometry is 1.7 moles bound per mole of enzyme in the ternary complex. The k cat values for the native and mutant enzymes were calculated this way and are shown in Table 1. As depicted in Figure 4, the interaction of substrates with L casei TS is "Ordered Bi Bi", with dUMP binding fIrst (Danenberg and Danenberg, 1978). The pertinent kinetic expressions as derived by the method of net rate constants (Cleland, 1975) are given in Equations 1-4. Equation 1 gives the expression for kcaJK~UMP at low CH2HJoiate concentration. k cat (1) At saturating CH 2HJolate, Equation I reduces to Equation 2. k cat (2) Sung-Woo Cho & Soo Young Choi Vol. 2 (1992) k \ [dUMP] TS .:;;;..;:::::::::=~ TS-dUMP .. *2 ,.1J " (C~" TS-dUMP - CHz Ii. folate ,••• ( ks [= kcarl .. Figure 4. Interaction of thymidylate synthase (fS) with substrates dUMP and CH 2HJoiate Equation 3 gives the expression for kcaJK~H2H4folate at low dUMP. (3) At saturating dUMP, Equation 3 reduces to Equation 4. ~+ks (4) In case of the active mutants, the Km values for dUMP were increased about lO-12-fold for the Tyr and Lys mutants, and about 2-fold for the Thr mutant. The Km values for CH2HJoiate did not significantly change in the Thr mutant, but was some 2-3-fold higher with the Tyr and Lys mutants. For inactive mutants (Val and Ser), the Km values icreased 6O-100-fold for dUMP and SS-70-fold for CH2HJolate. The Km values of the rest of the inactive mutants were too low to measure. For all active mutants, kca\ values were about 2 S-I, or 45% of the wild type. However, kca\ values of the inactive mutants were only 3 and 7% of the wild type for Val and Ser, respectively. The k cat values for mutations of Arg-178 were in all cases lower than those observed with this method for the wild type. Lys, Thr, and Tyr are optimal substitutions in terms of k cat being only 2-3 times lower than for the wild type. Most interesting is the kcaJKm value. For dUMP, the kcaJKm at saturating CH2HJoiate of the Thr mutant decreased 7-fold, whereas the Lys and Tyr mutants decreased 28- and 24-fold. For CH2HJolate, the kcaJKm values at saturating dUMP for the Lys and Tyr mutants decreased 6-8-fold, whereas for the Thr mutant kcaJKm decreased only 2-fold. The kcaJKm values of the inactive mutants were undetectable or much lower than those of the wild type. Discussion Our current understanding of the structure and catalytic activity of TS has been the result of biochemical study of many years. The Arg residues conserved at positions 23, 178, 179, and 218 form a positively charged binding surface for the 5' -phosphate of dUMP. Five of the guanidineoNH groups are within appropriate distance (2.5-3.2 A to provide hydrogen bonding or electrostatic stabilization of the phosphate anion of dUMP. Arg-178 is completely conserved in 113 all TS' sequenced to date (Bzilk et al., 1987), indicating an important role in structure/function of the enzyme. There are several features of the steady state rate equations for TS which deserve emphasis. At saturating concentrations of CH 2HJolate, kcaJKm for dUMP is a measure of the rate of association (k l ) of dUMP with free enzyme (Eq.' 2); thus, Km values for dUMP reflect kcaJkl . In contrast, at saturating dUMP concentrations kcaJKm values for CH2HJoiate are a combination of rate constants [k-Jcs/(k 4 + k s)J (Eq. 3 and 4) and thus Km values of CH 2HJoiate can be considered to be apparent binding constants. Since ok cat values are similar for the active mutants and only 3-fold lower than the wild type, once substrates are bound within the ternary complex the catalytic steps of the reaction are only moderately affected by the nature of the residue at 178. The most revealing effect of the mutations is on the net rate of asso~iation of dUMP with the enzyme (k l); this represents a step of the reaction pathway which can be readily measured as kcaJKm of dUMP at saturating CH2HJoiate (Eq. 2). According to the X-ray structure of the TS-Pi complex and molecular modeling (Hardy et al., 1987), suggesting that a conformational change of the protein occurs to accommodate binding of dUMP, and pre-steady state kinetic studies of the wild type enzyme (Mittelstaedt and Schimerlik, 1986), the first rapid formation of a weak complex (~~ 300 mM), is followed by a slower isomerization (k~ 300 S- I). The net rate constant calculated from these data (-1 ~ - IS- I) is in good agreement with the value of kl calculated from steady state kinetics, suggesting that the same process is being measured. It is proposed that the isomerization step seen in pre-steady state kinetics reflects the anticipated conformational change. The kl values obtained from steady state kinetics must primarily reflect the slow step of the interaction, and thus provide a kinetic probe for the conformational change. As shown in Table 1, kcaJKm of CH 2HJoiate at saturating dUMP is not very different in the Thr mutant. Since this parameter measures [k-Jcs(k 4 + k s)J, the Thr mutant does not significantly differ in any of the steps after formation of the binary TS' dUMP complex. In contrast, kcaJKm values of CH2HJoiate for the Lys and Tyr mutants are reduced 6-8-fold compared to wild type TS. Since kcat values of Tyr and Lys mutants decreased only 2-fold compared to the wild type, and the apparent Km of CH2HJoiate increased, the effects can be attributed to binding of the cofactor. It is not apparent why the active mutants would show the different effects, but it is unusual among those studied in that it significantly affects both the rate of association of dUMP and binding of CH2HJolate. Although the differential effects of the Arg-178 mutants have allowed the aforementioned interpretations, it is apparent that although Arg-178 is bound to the phosphate of dUMP, it is not essential for substrate binding or catalytic activity. However it can be replaced by Lys, Thr, and Tyr amino acids without drastic changes in its biological activity. 0 114 Mutagenesis of Arg-178 in Thymidylate Synthase How do we explain the enigma of the Arg-178 which is bound to the 5'-phosphate of dUMP, but is not essential for activity? Our favored explanation is that "plasticity" of the protein, and compensatory function of other residues are important factors in the retention of high activity of the 178 mutants. The guanidinium groups of Arg-23, Arg-179, and Arg-218 are also bound to Pi (Hardy et al., 1987) or the 5' -phosphate of dUMP; it is proposed that upon mutation of Arg-178 to a residue that cannot bind phosphate, its important functions are transferred to the other Arg residues. Studies on the mutagenesis of these other Arg residues of TS are in progress in our laboratory. A second enigma resides in the fact that Arg-178 is conserved in all TS' thus far sequenced (from bacteria, bacteriophage, protozoa, yeast, and mammals); yet, it is not essential, or even significantly advantageous, for binding or catalysis. This feature has been observed with other conserved residues which are also apparently important to structure/function (Maley et al., 1990; Dev et aI., 1989; Clirnie and Santi, 1990). We surmise that conserved amino acid residues of TS may serve crucial roles which are not revealed from such studies. The approach we have used has several practical advantages. (1) A synthetic gene with conveniently placed, unique restriction sites permits mutagenesis at high mutagenesis efficiency. (2) A mutagenesis vector which also serves as a high expression vector avoids further subcloning. (3) A selection system for the desired property permits rapid identification of mutants of interest. This combination of genetic and biochemical techniques could be used to address a broad range of questions relating to the structure and function of the enzyme. Adequate characterization of the corresponding mutant proteins would involve kinetic studies of purified mutants. Acknowledgments We thank Dr. Daniel V. Santi for his generous gift of synthetic TS gene (PSCTS9) and oligonucleotides. This work was supported in part by grants from Korea Science and Engineering Foundation (913-0401013-2) and the Asan Institute for Life Sciences to S.W. Cho (90-09-035). Mol. Cells References Belfort, M., Maley, G. F., and Maley, F. (1980) A rch. Biochem. Biophys. 204, 340-349 Bruice, T. W., and Santi, D. V. (1982) Biochemistry 21, 6703-6709 Bzik, D. J., L~ W. R , Horii, T., and Inselberg, J. (1987) Proc. Natl. Acad. Sci. USA 84, 8360-8364 Cipollo, K L., and Dunlap, R R (1979) Biochem istry 18, 5537-5541 Cipollo, K L., Lewis, C. A , Jr., Ellis, P. D., and Dunlap, R R (1982) J Bioi. 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(1988) DNA 7, 235-241 Pogolotti, A L., Jr., Danenberg, P. v., and Sant~ D. V. (1986) J Med. Chern. 29, 478-482 Read, S. M., and Northcote, D. H. (1981) Ana!. Biochem. 116, 53-64 Reidhaar-Olson, J., and Sauer, R (1988) Science 241, 53-57 Santi, D. v., and Danenberg, P. (1984) in Chemistry and Biochemistry of Folates (Blakely, R , and Benkovic, S., eds) pp. 345-398, John Wiley & Sons, New - York Santi, D. v.. McHenry, C. H., Raines, R T., and Ivanetich, K I. (1987) Biochemistry 26, 8606-8613