Proc. Nati. Acad. Sci. USA
Vol. 73, No. 6, pp. 1848-1852, June 1976 Biochemistry
Amino acid sequence at the FdUMP binding site of thymidylate synthetase (cyanogen bromide peptides/cysteine)
RONALD L. BELLISARIO, GLADYS F. MALEY, JOHN H. GALIVAN, AND FRANK MALEY Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201
Communicated by Saul Roseman, March 11, 1976
Cyanogen bromide treatment of thymidylate ABSTRACT synthetase of Lactobacillus casei, which had been converted to a ternary complex with [2-4CJFdUMP and 5,10-methylenetetrahydrofolate followed by Scarboxymethylation, yielded at least our visible peptide bands, the largest with a molecular weight of about 13,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea. Identical results were obtained with enzyme that had all four of its cysteinyl residues S-carboxymethylated with iodo[1-14C]acetate in the absence of FdUMP and cofactor. In each case, only the second band from the top of the gel (CN2), with an approximate molecular weight of 10,000, was labeled. Analysis of CN2 that had been labeled with [2-4C]FdUMP and nonradioactive iodoacetate and of that labeled only with iodo[1-'4C]acetate revealed that their amino-acid contents were almost identical except for the presence of two S~carboxymethy(Cm) cysteinyl residues in the latter eptide and only one in FdUMP-CN2. A nonapeptide was isoIated from (Cm)2-CN2 after chymotrypsin digestion that contained the following sequence by dansyl-Edman analysis: AlaLeu-Pro-Pro[Cm-CysJHis-Thr-Leu-Tyr. This peptide was found to be located on the NH2-terminal end of CN2. Automatic sequence analysis of the first 13 residues of (Cm)2-CN2 and of the FdUMP-containing CN2 yielded identical results except for the fifth, or cysteinyl, residue, which could not be identified in the latter peptide. These findings strongly suggest that FdUMP is linked to a cysteinyl residue in thymidylate synthetase that has been inactivated irreversibly by this nucleotide. Since the observation (1, 2) that FdUMP inhibits thymidylate synthetase by binding to a region believed to be the active site, numerous studies have been directed toward unraveling the nature of this site. Initial attempts were hampered by the lack of adequate quantities of enzyme until a method was perfected for obtaining crystalline enzyme from methotrexate- and dichloromethotrexate-resistant strains of Lactobacillus casei (3, 4). Studies with sulfhydryl inhibitors (3, 5, 6) suggest that at least one of the enzyme's four cysteinyl residues reacts with FdUMP by adding across the 5,6-double bond of its pyrimidine ring. Although model studies favoring a nucleophilic attack at the 6-position of the pyrimidine ring have been reported (7, 8), the role of cysteine as a nucleophile (9) was questioned recently by Sommer and Santi (10) when they could not obtain evidence for cysteine in an FdUMP-containing peptide isolated from the synthetase. The latter finding can be questioned, in turn, as a result of prior studies with sulfhydryl inhibitors (3, 5, 6), which implicated a sulfhydryl group at the active site and more recent ones with a5S-labeled thymidylate synthetase (11). In an attempt to resolve the question of whether or not cysAbbreviations: CN2, a second cyanogen bromide peptide on sodium
chymotryptic dodecyl sulfate-polyacrylamide gels; C2A and C2B,two-dimensional peptides from CN2 derivatives isolated by BioGel and paper chromatography; Cm, S-carboxymethyl; FdUMP, 5-fluorodeoxyuridine 5'-phosphate; 5,10-CH2H4 folate, 5,10-methylenetetrahydrofolate.
teine is associated with the FdUMP binding site, we have developed procedures for isolating two types of peptides, one containing an FdUMP and an S-carboxymethylated(Cm) residue (Cm,FdUMP)-CN2 and another containing two S-carboxymethylated residues, (Cm)2-CN2. Evidence will be presented demonstrating that these peptides are identical in amino-acid sequence and that FdUMP is most probably linked to cysteine.
MATERIALS AND METHODS Materials. Trimethylamine and HI were purchased from Eastman. Chen Ching polyamide plates were obtained from the Gallard-Schlesinger Chemical Mfg. Corp., microcrystalline cellulose plates from Brinkmann, and aluminum-backed silica gel plates (EM Labs) from VWR Scientific. SP-Sephadex, C-25, was purchased from Pharmacia and leucine aminopeptidase and a-chymotrypsin from the Worthington Biochemical Corp. Bovine carboxypeptidase A was a gift from Dr. T. H. Plummer, Jr., of this Division. Sequencer grade reagents were obtained from Beckman Instruments and Pierce Chemicals. [1-14C]- and [3H]iodoacetate were purchased from the New England Nuclear Corp. and [2-'4C]fluorodeoxyuridine from Schwarz/ Mann. The nucleoside was converted to FdUMP with POC13 by a micromodification of the procedure of Yoshikawa et al. (12). Thymidylate synthetase was purified and crystallized as described by Galivan et al. (13). Preparation of Thymidylate Synthetase Containing Four Groups. Thymidylate synthetase (135 S_[14CjCarboxymethyl mg, 1.9 ,mol) was dissolved in 5 ml of a solution containing 6 M guanidine-HCl, 1 M Tris-HCl (pH 8.0), and 0.1 M 2-mercaptoethanol. The enzyme solution was incubated for 4 hr at room temperature under a nitrogen atmosphere and then reacted for 10 min with 8.5 mg of iodo[1-14C]acetate (27.2 .Ci/mg). After this period, 94 mg of unlabeled iodoacetate were added and the incubation was continued for another 10 min. 2-Mercaptoethanol (35 ,ul) was then added, and the resulting solution was dialyzed in the cold against four changes of 6 liters each of 5% acetic acid. Amino-acid analyses were used to determine the extent of S-carboxymethylation. Preparation of Synthetase with One S{14C]Carboxymethyl Group. Thymidylate synthetase (5 mg, 72 nmol) was passed through a Sephadex G-25 column (1 X 25 cm) with 0.05 M potassium phosphate (pH 7.0) to remove 2-mercaptoethanol. The enzyme was then treated at room temperature with iodo[ I 14C]acetate (7.2 iAmol, 3000 cpm/nmol) in 2.0 ml of 0.1 M Tris.HCI (pH 8.0) under an argon atmosphere. After 60 min it was 96% inhibited, even when assayed in the presence of excess 2-mercaptoethanol. From 0.95 to 1.1 mol of iodoacetate was incorporated per mol of enzyme, as determined by a nitrocellulose assay (2). The carboxymethylated enzyme was dialyzed against three changes of 2 liters each of 0.1 M 1848
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Bellisario et al. NH4HCO3 containing 2-mercaptoethanol for a total of 18 hr. The residual unreacted cysteinyl residues were then carboxymethylated with unlabeled iodoacetate (14). Preparation of [2-14C]FdUMP-Enzyme Ternary Complex. One hundred milligrams (1.4 imol) of thymidylate synthetase were dissolved in 5 ml of 0.1 M 2-mercaptoethanol and dialyzed for 16 hr against two 1-liter changes of 0.05 M potassium phosphate (pH 7.0) and 0.02 M 2-mercaptoethanol. The dialyzed enzyme, with a specific activity of 3.4 units/mg of protein (3), was converted to a ternary complex by incubating with 8 gmol of L-5,10-CH2H4 folate and 3.3 ,umol of [214C]FdUMP (3 X 106 cpm/gmol) at 300 for 150 min in the dark. The complexed enzyme was S-carboxymethylated with unlabeled iodoacetate as described above and dialyzed against three 4-liter changes each of 10% acetic acid and 5 mM 2mercaptoethanol, followed by lyophilization. According to the nitrocellulose filter assay (2), 2.2 mol of [2-14C]FdUMP per mol of enzyme had been incorporated, and of this, 72% (1.6 mol of FdUMP per mol of enzyme) was still bound after dialysis. Isolation of Labeled Peptides After Cyanogen Bromide Treatment. The [14C]Cm- and [2-14C]FdUMP-enzyme complexes were converted to peptides with cyanogen bromide by the procedure of Steers et al. (15). The extent of cleavage was followed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea (16) and by amino-acid analysis for methionine. The peptides were separated on an SP-Sephadex column by a modification of the procedure of Langley et al. (17) (Fig. 1). Amino Acid and Sequence Analysis. Peptides were hydrolyzed with 6 M HC1 at 110° for 22 hr in evacuated, sealed tubes. To minimize destruction of Cm-cysteine and tyrosine (18), 1 ,ul of thioglycolic acid and 20 ,l of a 5% solution of phenol were added prior to hydrolysis. Single column amino-acid analysis was performed on a model 5AH amino-acid analyzer (Jeol Co.) with Durrum DC-lA resin and pico-buffers. Manual sequencing with 10 nmol of peptide was performed by a modification (19) of the dansyl-Edman procedure (20). The coupling buffer contained 200 ,ul of trimethylamine (25% aqueous solution)-pyridine-water (0.5:4:2, vol/vol) and 5 ,l of phenylisothiocyanate. Dansyl amino acids were identified by thin-layer chromatography on polyamide sheets (5 X 5 cm) (21). Automatic peptide sequencing was performed with a Beckman model 890B sequencer using the 0.1 M Quadrol peptide program of Brauer et al. (22). The amino-acid phenylthiohydantoins were identified by a combination of thinlayer chromatography on silica gel sheets (23, 24), and isothermal gas-liquid chromatography (25). Hydrolysis of Peptides with Leucine Aminopeptidase and Carboxypeptidase A. Samples of peptide (10 nmol) were hydrolyzed for 21 hr at 370 with leucine aminopeptidase (0.2 nmol) in 0.1 ml of 0.1 M Tris-HCl (pH 8.6) containing 2.5 mM MgCl2. The reaction was terminated by the addition of 10Qul of 1 M HCI and 0.9 ml of Durrum pico-buffer A. The resultant solution was applied immediately to the amino-acid analyzer. For carboxypeptidase A treatment, 10 nmol of peptide was hydrolyzed for 24 hr at 25° with 0.2 nmol of enzyme in 0.1 ml of 0.1 M potassium phosphate (pH 8.2). The hydrolysis products were subjected to amino-acid analysis as described for leucine aminopeptidase. Limited carboxypeptidase A digestions were conducted at 250 with 0.6 nmol of peptide and 0.6 pmol of enzyme. At intervals of 15, 30, and 60 min, aliquots containing 0.2 nmol of peptide were removed and frozen immediately in liquid nitrogen. After Iyophilization, the hydrolysis products were dansylated and identified by thin-layer chromatography
1849
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FIG. 1. SP-Sephadex chromatography of cyanogen bromide peptides from (Cmj2-14CJFdUMP)2- or (["C]Cm)4-thymidylate synthetase. The cyanogen bromide peptides from 100 mg of enzyme were dissolved in 0.01 M ammonium formate (pH 2.8) containing 6 M urea and passed through an SP-Sephadex column (1.5 X 25 cm), which was then washed with 100 ml of the ammonium formate-urea solution. The column was next eluted (A to B) with a linear gradient of 0-1 M NaCl, with each reservoir containing 300 ml of the 0.01 M ammonium formate-6 M urea. On completion of the gradient, the labeled CN2 peptide (shaded area) was eluted from the column with 0.5 M NH4OH-6 M urea (C). Urea was removed from the pooled peptide fractions by dialysis against 30% acetic acid in Spectropor 3500 dialysis tubing (Spectrum Medical Industries).
on polyamide plates. Appropriate enzyme blanks were included for each proteolytic reaction. Isolation of Radioactive Chymotryptic Peptides from [14CJCm-CN2. To approximately 90 nmol of (['4C]Cm)2-CN2 (9 X .105 cpm) was added a 10-fold excess of unlabeled peptide. The combined peptides (1.0 ,umol; 9 X 105 cpm) were suspended in 3 ml of 1% NH4HCO3 containing 0.1 mg of chymotrypsin. After 6 hr at 370 the suspension cleared, and 0.1 mg more of enzyme was added. Hydrolysis was continued for a total of 16 hr at 370, at which time the solution was lyophilized. The residue was dissolved in 1 ml of 10% acetic acid and fractionated on a column of BioGel P-2 (1.5 X 97 cm), with further purification of the radioactive peptides by two-dimensional ascending chromatography on Whatman 3MM paper. Solvent 1 was n-butanol-acetic acid-water (4:1:5, vol/vol); solvent 2, n-butanol-20% pyridine acetate (pH 5.4)-water (1:1:1, vol/vol). The 14C-labeled peptides, detected by radioautography with Kodak RP/R2 x-ray film, were eluted from the paper with 20% pyridine acetate (pH 5.4). To assess the purity of the peptides, analytic two-dimensional mapping on cellulose thin-layer plates (20 X 20 cm) was carried out as described by Schachner and Zillig (26). Nonradioactive peptides were detected with 0.2% ninhydrin in acetone.
RESULTS Cyanogen bromide cleavage of thymidylate synthetase. In general agreement with the findings of Loeble and Dunlap (27), four distinct bands and one or two less visible bands were obtained when the cyanogen bromide-treated carboxymethylated enzyme was electrophoresed in polyacrylamide gel containing urea and sodium- dodecyl sulfate. The largest band (CN1) had a molecular weight of about 13,000; the smallest
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Biochemistry: Bellisario et al.
Proc. Nati. Acad. Sci. USA 73 (1976)
Table 1. Comparison of amino-acid content of peptide derivatives from thymidylate synthetase
(Cm, FdUMP)2Synthetase (residues/70,000 g)
Amino acid
(Cm)4-Synthetase
Cm-Cys Asp Thr Ser Glu Pro Gly Ala Val Met Ile
3.54 71.2 31.4 23.2 56.6 30.2 37.1 43.4 26.6 11.9 25.9 68.7 27.8 38.0 35.5 35.7 23.5
Leu Tyr Phe Lys His Arg
(Cm, FdUMP)CN2 (residues/10,000 g)
(Cm)2-CN2
1.97 71.8 34.2 21.2 56.0 31.4 36.7 43.4 26.2 12.0 25.8 67.5 27.4 38.4 36.2 37.0 24.6
1.26 10.9 4.01 3.59 9.20 5.98 4.85 6.05 3.52
C2A
C2B (residues)*
0.87 10.8
1.0
1.09
4.09
1.04
0.85
3.49 9.44 6.04 5.16 6.31 4.08
3.60 1.98 2.44 0.91 2.18
0.97
1.92
2.26 1.03
0.48t
0.49t 3.64 12.6 4.09 5.71 2.89 5.68 2.70
4.10 12.2 4.09 5.49 2.85 5.66 2.78
0.97 1.90
0.97
* Mol/mol of peptide. t Based on recovered homoserine lactone.
band was about 2000, but because bands of this size and smaller tended to elute from the gel on destaining it was difficult to determine the number of such bands. The sum of the calibrated molecular weights of the peptide bands approximated 35,000, in good agreement with that reported for a subunit of the L. casei thymidylate synthetase (3). When the native enzyme was labeled with radioactive FdUMP, iodoacetate, or N-ethylmaleimide and then S-carboxymethylated under denaturing conditions with unlabeled iodoacetate, only the second band (CN2) was found to be radioactive. Since the enzyme is composed of two identical subunits (27), CN2 with a molecular weight of 10,000 appears to account for the two cysteines in each subunit (Table 1). It is of interest to note that carboxymethylation of the FdUMP-tre4ted enzyme yielded only two of the four Cm-cysteinyl residues usually obtained with untreated enzyme. Chymotryptic peptides from CN2 Initially, ([14C]Cm)2-CN2 was isolated by pooling selected fractions after BioGel P-10 column (1.5 X 96 cm) chromatography. This peptide was then digested with chymotrypsin; a thin-layer, two-dimensional chromatogram is presented in Fig. 2A. On BioGel P-2 chromatography of the digest, two radioactive peaks were eluted, Cm-C2A and Cm-C2B. The smaller of the two, Cm-C2B, was purified to homogeneity by two-dimensional paper chromatography (Materials and Methods) and migrated to the shaded region designated by the arrow in Fig. 2A. Amino-acid analysis of Cm-C2B after acid hydrolysis (Table 1) or leucine aminopeptidaset digestion yielded similar results, with the most significant finding being the presence of one Cm-cysteinyl residue per mol of peptide. This value represents about half that in (Cm)2-CN2. The isolated labeled C2B from ([14C]Cm)2-CN2 was analyzed by a manual dansyl-Edman procedure (18) to yield the sequence: Ala-Leu-Pro-Pro-(Cm-Cys)-His-Thr-Leu-Tyr Verification of the sequence at the carboxyl-terminal end was obtained with carboxypeptidase A; tyrosine and leucine t
Normally leucine aminopeptidase does not hydrolyze proline bonds, but this preparation was apparently contaminated with prolidase.
were released quantitatively but threonine and histidine were hydrolyzed to a lesser extent (30% and 10%, respectively). Recent studies by us, where one mol of iodo[l-'4C]acetate was fixed to one mol of enzyme to completely inactivate it, followed by unlabeled iodoacetate to carboxymethylate the remaining sulfhydryl groups, yielded a "4C-labeled chymotryptic peptide from (Cm)2-CN2 with half the specific activity of the same peptide from synthetase that had been completely S-carboxymethylated with iodo[l-14C]acetate. The radioactive peptide migrated to the same region as that occupied by Cm-C2B (Fig. 2A, arrow). The nature of the proposed reaction is indicated below: two CN2 peptides are presented to represent the two identical subunits of the synthetase, with only one of the native enzyme's four sulfhydryl groups being labeled. Cm Cm Ala fiCN2-
1
_I
["4CJCm
Cm Chymotrypsin
Cm
Cm
Ala-C2B--Tyr Ala-I-C2B-Tyr [14CJCm
+ peptides +
I
-C2A
I- -C2A
-
t Cm
After chymotrypsin treatment of (Cm,[2-"4C]FdUMP)CN2, two-dimensional chromatographic analysis indicated that the ninhydrin spot corresponding to Cm-C2B was missing (Fig. 2B, arrow) and that the radioactivity was located in a region other than that shown in Fig. 2A. This finding strongly suggests that FdUMP was present in place of one of the two Cm-residues of (Cm)2-CN2, most probably that in Cm-C2B, and is supported by the amino-acid data in Table 1 and the sequence analyses below. Isolation and sequence analysis of FdUMP- and Cm-. cysteine-containing CN2 To facilitate the isolation of the cyanogen bromide peptides, we used SP-Sephadex column chromatography described in
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Bellisario et al.
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1851
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FIG. 2. Two-dimensional thin-layer chromatography of a chymotryptic digest of (A) ([14C]Cm)2-CN2 and (B) (Cm,[2-14C]FdUMP)-CN2. The arrows designate the locations of the Cm-cysteine-containing peptide, C2B. The stippled regions represent those of relatively intense radioactivity and the dotted regions, those of less intensity. Solvents 1 and 2 are described in Materials and Methods.
the legend of Fig. 1. Under these conditions, both the ([14C]Cm)2- and the (Cm,[2-14C]FdUMP)-CN2 peptides were the most tightly bound, but could be released with 0.5 M NH4OH-6 M urea after the gradient elution procedure. As indicated in Table 1, the two peptides are almost identical in amino-acid composition, except for the number of Cm-cysteinyl residues, which in the case of the (Cm,FdUMP)-CN2 was always less than 1. What happens to the second cysteinyl residue on acid hydrolysis of (Cm,FdUMP)-CN2 is not known, but this fact, plus others to be discussed, indicates that the FdUMP-cysteine linkage does not yield cysteine on acid hydrolysis. Although the Cm-Cys value in (Cm)2-CN2 is closer to 1 than to 2 in this particular preparation, others have yielded values closer to 1.5. The low values for Cm-Cys are not surprising in view of the instability of this residue. More representative data, however, were obtained with the isolated chymotryptic peptides, Cm-C2A and Cm-C2B, as indicated by the amino-acid analyses in Table 1. Sequence analysis of Cm- and FdUMP-labeled CN2 Because the amino-acid compositions of (Cm)2-CN2 and (Cm,FdUMP)-CN2 were so similar, an effort was made to determine the location of the chymotryptic peptide C2B, in the 90-100 amino acids of CN2. Preliminary dansyl-Edman analysis of CN2 revealed that the nonapeptide, C2B, might be located on the amino-terminal end because a sequence of Ala-Leu-Pro was obtained. This fortunate coincidence was confirmed by automatic sequence analysis of the first 13 residues from each of the two peptides: (Cm,FdUMP)-CN2: Ala-Leu-Pro-Pro-
[X]-His-Thr-Leu-Tyr-Gln-Phe-Tyr-Val(Cm)2-CN2: Ala-Leu-Pro-Pro-
[Cm-Cys]-His-Thr-Leu-Tyr-Gln-Phe-Tyr-ValAlthough the association of radioactivity with the fifth residue of (Cm)2-CN2 confirmed the presence of cysteine at this position, comparable results were not obtained with (Cm,[214C]FdUMP)-CN2. In this case, radioactivity was obtained in several residues, particularly in the early ones, indicating that the acid conditions associated with cleavage were effecting a slow release of FdUMP. DISCUSSION Although most investigators generally agree on the nature of the ternary complex resulting from the interaction of thymi-
dylate synthetase with 5,10-CH2H4 folate and FdUMP, some question has arisen concerning the nucleophile that initiates complex formation. Danenberg et al. have proposed (5), because of the decrease in absorbance at 270 nm, that a cysteine sulfhydryl saturates the 5,6-double bond of the pyrimidine ring of FdUMP. This in turn is believed to promote a methylene bridge structure between 5,10-CH2H4 folate and the 5-position of the enzyme-FdUMP complex (5, 28), as originally proposed by Friedkin and Kornberg (29). The studies of Santi and coworkers, however, although in general agreement with the ternary complex thesis, are not in harmony with respect to the nucleophile. Thus, their studies with N-ethylmaleimide (30), in which 4 mol were found to react with the FdUMP-enzyme complex, and their isolation of an FdUMP-peptide (10) containing [Thr, His, Ala, Leu, (Pro)2], have led them to propose threonine or histidine as the nucleophile. This peptide is probably a smaller version of C2B isolated by us. Studies by others with sulfhydryl reagents, however, have shown that the synthetase can be completely inhibited by pchloromercuribenzoate (3), N-ethylmaleimide (3), iodoacetamide (6), and showdomycin (31), and can be protected by the substrate, dUMP (5). It appears that reaction of only one of the enzyme's four sulfhydryl groups is sufficient to effect complete inhibition (3, 6). Similar to the findings of Dunlap et al. (3) with p-chloromercuribenzoate, we obtained identical results with N-ethylmaleimide and other compounds to be reported. In addition, by amino-acid analyses of the products of the reaction of N-ethylmaleimide with the FdUMP-enzyme complex under conditions similar to those described by McHenry and Santi (30), we have found that sufficient amino acids other than cysteine react to account for 4 mol of N-ethylmaleimide per mol of FdUMP-enzyme complex. When the number of Nsuccinyl cysteinyl residues from FdUMP-treated synthetase was compared with that from untreated enzyme, approximately two fewer residues were found in the treated enzyme, indicating that FdUMP had protected two of the enzyme's four sulfhydryl groups from N-ethylmaleimide. Similar results, but with less nonspecific reaction, were obtained on iodoacetate treatment of the FdUMP-enzyme complex under denaturing conditions. Recent studies (11) with 3SS-labeled enzyme are also in agreement with the presence of cysteine at the FdUMP reactive site.
The reason for the apparent absence of cysteine in the acid-hydrolyzed FdUMP-peptide obtained by Sommer and Santi (10) and by ourselves is not apparent at present, although
1852
Biochemistry: Bellisario et al.
there is no reason to assume that an FdUMP-cysteine adduct should yield cysteine on acid hydrolysis. Alternatively, it should not be ruled out that the peptide isolated by them does not contain cysteine, but is coincidently similar in amino-acid composition to the one isolated by us. It is difficult, however, to deny an association of FdUMP with Cys5 of CN2, based on the sequence analyses presented for (Cm,FdUMP)-CN2 and (Cm)2-CN2 and the amino-acid data of Table 1. These data are consistent with the less drastic conditions of leucine aminopeptidase hydrolysis, where the other proposed nucleophiles (10), threonine and histidine, could be accounted for in both peptides. The above results indicate that FdUMP and cysteine are linked in some, as yet, undefined manner. It should be emphasized that these findings do not imply that cysteine is the nucleophile that initiates catalysis at the active site or that dUMP and FdUMP react identically at this site. Although logic dictates that this may be the case because of the structural similarities of dUMP and FdUMP, other factors must also be considered. Thus, although FdUMP reacts initially as a competitive inhibitor of dUMP, the reaction does become noncompetitive with time (32-34) and FdUMP eventually becomes covalently bound to two sites on the enzyme. In contrast, recent circular dichroism (6) and binding studies (35) with dUMP indicate that dUMP binds primarily to a single site on the synthetase, in agreement with the inhibition experiments with sulfhydryl reagents. Whether dUMP and FdUMP react initially at the same site, and FdUMP becomes covalently bound to that site or is transferred subsequently to another site where it becomes irreversibly bound, remains to be determined. We thank Don U. Guarino for his excellent technical assistance and Dr. B. Pollara of the Kidney Disease Institute for permitting us the use of his sequenator. This research was supported in part by Public Health Service Grant GM-20371 from the National Institute of General Medical Sciences, PHS/DHEW. 1. Langenbach, R. J., Danenberg, P. V. & Heidelberger, C. (1972) Biochem. Biophys. Res. Commun. 48,1565-1571. 2. Santi, D. V. & McHenry, C. S. (1972) Proc. Natl. Acad. Sci. USA 69, 1855-1875. 3. Dunlap, R. B., Harding, N. G. L., & Huennekens, F. M. (1971) Biochemistry 10, 88-97. 4. Leary, R. P. & Kisliuk, R. L. (1971) Prep. Biochem. 1, 47-54. 5. Danenberg, P. V., Langenbach, R. J. & Heidelberger, C. (1974)
Biochemistry 13,926-933.
6. Leary, R. P., Beaudette, N. & Kisliuk, R. L. (1975) J. Biol. Chem.
250,4864-4868. 7. Santi, D. V. & Brewer, C. F. (1968) J. Am. Chem. Soc. 90, 6236-6238.
Proc. Natl. Acad. Sci. USA 73 (1976) 8. Santi, D. V. & Brewer, C. F. (1973) Biochemistry 12,2416-2424. 9. Kalman, T. I. (1971) Biochemistry 10, 2567-2573. 10. Sommer, H. & Santi, D. V. (1974) Biochem. Biophys. Res. Commun. 57,689-695. 11. Danenberg, P. V. & Heidelberger, C. (1975) Fed. Proc. 34,522. 12. Yoshikawa, M., Kato, T. & Takenishi, T. (1967) Tetrahedron Lett. 50,5065-5068. 13. Galivan, J., Maley, G. F. & Maley, F. (1975) Biochemistry 14, 3338-3343. 14. Kuhn, R. W., Walsh, K. A. & Neurath, H. (1974) Biochemistry 13,3871-3877. 15. Steers, E., Jr., Craven, G. R., Anfinsen, G. B. & Bethune, J. L. (1965) J. Biol. Chem. 240,2478-2484. 16. Swank, R. T. & Munkres, K. D. (1971) Anal. Biochem. 39, 462-477. 17. Langley, K. R., Fowler, A. V. & Zabin, I. (1975) J. Biol. Chem. 250,2587-2592. 18. Howard, S. M. & Pierce, J. G. (1969) J. Biol. Chem. 244, 6468-6476. 19. Bellisario, R., Carlsen, R. B. & Bahl, 0. P. (1973) J. Biol. Chem. 248,6796-6809. 20. Gray, W. R. (1967) in Methods in Enzymology, eds. Hirs, C. H. W. & Timasheff, S. N. (Academic Press, New York), Vol. 25, part B, pp. 333-344. 21. Bruton, C. J. & Hartley, B. S. (1970) J. Mol. Biol. 52, 165-178. 22. Brauer, A. W., Margolies, M. N. & Haber, F. (1975) Biochemistry 14,3029-3035. 23. Inglis, A. S. & Nichols, P. W. (1973) J. Chromatogr. 79,344-346. 24. Summer, M. R., Smythers, G. W. & Oroszlan, S. (1973) Anal. Biochem. 53,624-628. 25. Niall, H. D. (1973) in Methods in Enzymology, eds. Hirs, G. H. W. & Timasheff, S. N. (Academic Press, New York), Vol. 27, part D, pp. 942-1010. 26. Schachner, M. & Zillig, W. (1971) Eur. J. Biochem. 22,513-519. 27. Loeble, R. B. & Dunlap, R. B. (1972) Biochem. Biophys. Res. Commun. 49, 1671-1677. 28. Santi, D. V., McHenry, C. S. & Sommer, H. (1974) Biochemistry 13,471-481. 29. Friedkin, M. & Kornberg, A. (1957) in A Symposium on the Chemical Basis of Heredity, eds. McElroy, W. D. & Glass, B. (Johns Hopkins University Press, Baltimore, Md.), pp. 609-614. 30. McHenry, C. S. & Santi, D. V. (1974) Biochem. Biophys. Res. Commun. 57,204-208. 31. Kalman, T. I. (1972) Biochem. Biophys. Res. Commun. 49, 1007-1013. 32. Blakley, R. L. (1963)J. Biol. Chem. 238,2113-2118. 33. Mathews, C. K. & Cohen, S. S. (1963) J. Biol. Chem. 238,367370. 34. Lorenson, M. Y., Maley, G. F. & Maley, F. (1967) J. Biol. Chem.
242,3332-344.
35. Galivan, J., Maley, G. F. & Maley, F. (1976) Biochemistry 15, 356-362.
Vol. 73, No. 6, pp. 1848-1852, June 1976 Biochemistry
Amino acid sequence at the FdUMP binding site of thymidylate synthetase (cyanogen bromide peptides/cysteine)
RONALD L. BELLISARIO, GLADYS F. MALEY, JOHN H. GALIVAN, AND FRANK MALEY Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201
Communicated by Saul Roseman, March 11, 1976
Cyanogen bromide treatment of thymidylate ABSTRACT synthetase of Lactobacillus casei, which had been converted to a ternary complex with [2-4CJFdUMP and 5,10-methylenetetrahydrofolate followed by Scarboxymethylation, yielded at least our visible peptide bands, the largest with a molecular weight of about 13,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea. Identical results were obtained with enzyme that had all four of its cysteinyl residues S-carboxymethylated with iodo[1-14C]acetate in the absence of FdUMP and cofactor. In each case, only the second band from the top of the gel (CN2), with an approximate molecular weight of 10,000, was labeled. Analysis of CN2 that had been labeled with [2-4C]FdUMP and nonradioactive iodoacetate and of that labeled only with iodo[1-'4C]acetate revealed that their amino-acid contents were almost identical except for the presence of two S~carboxymethy(Cm) cysteinyl residues in the latter eptide and only one in FdUMP-CN2. A nonapeptide was isoIated from (Cm)2-CN2 after chymotrypsin digestion that contained the following sequence by dansyl-Edman analysis: AlaLeu-Pro-Pro[Cm-CysJHis-Thr-Leu-Tyr. This peptide was found to be located on the NH2-terminal end of CN2. Automatic sequence analysis of the first 13 residues of (Cm)2-CN2 and of the FdUMP-containing CN2 yielded identical results except for the fifth, or cysteinyl, residue, which could not be identified in the latter peptide. These findings strongly suggest that FdUMP is linked to a cysteinyl residue in thymidylate synthetase that has been inactivated irreversibly by this nucleotide. Since the observation (1, 2) that FdUMP inhibits thymidylate synthetase by binding to a region believed to be the active site, numerous studies have been directed toward unraveling the nature of this site. Initial attempts were hampered by the lack of adequate quantities of enzyme until a method was perfected for obtaining crystalline enzyme from methotrexate- and dichloromethotrexate-resistant strains of Lactobacillus casei (3, 4). Studies with sulfhydryl inhibitors (3, 5, 6) suggest that at least one of the enzyme's four cysteinyl residues reacts with FdUMP by adding across the 5,6-double bond of its pyrimidine ring. Although model studies favoring a nucleophilic attack at the 6-position of the pyrimidine ring have been reported (7, 8), the role of cysteine as a nucleophile (9) was questioned recently by Sommer and Santi (10) when they could not obtain evidence for cysteine in an FdUMP-containing peptide isolated from the synthetase. The latter finding can be questioned, in turn, as a result of prior studies with sulfhydryl inhibitors (3, 5, 6), which implicated a sulfhydryl group at the active site and more recent ones with a5S-labeled thymidylate synthetase (11). In an attempt to resolve the question of whether or not cysAbbreviations: CN2, a second cyanogen bromide peptide on sodium
chymotryptic dodecyl sulfate-polyacrylamide gels; C2A and C2B,two-dimensional peptides from CN2 derivatives isolated by BioGel and paper chromatography; Cm, S-carboxymethyl; FdUMP, 5-fluorodeoxyuridine 5'-phosphate; 5,10-CH2H4 folate, 5,10-methylenetetrahydrofolate.
teine is associated with the FdUMP binding site, we have developed procedures for isolating two types of peptides, one containing an FdUMP and an S-carboxymethylated(Cm) residue (Cm,FdUMP)-CN2 and another containing two S-carboxymethylated residues, (Cm)2-CN2. Evidence will be presented demonstrating that these peptides are identical in amino-acid sequence and that FdUMP is most probably linked to cysteine.
MATERIALS AND METHODS Materials. Trimethylamine and HI were purchased from Eastman. Chen Ching polyamide plates were obtained from the Gallard-Schlesinger Chemical Mfg. Corp., microcrystalline cellulose plates from Brinkmann, and aluminum-backed silica gel plates (EM Labs) from VWR Scientific. SP-Sephadex, C-25, was purchased from Pharmacia and leucine aminopeptidase and a-chymotrypsin from the Worthington Biochemical Corp. Bovine carboxypeptidase A was a gift from Dr. T. H. Plummer, Jr., of this Division. Sequencer grade reagents were obtained from Beckman Instruments and Pierce Chemicals. [1-14C]- and [3H]iodoacetate were purchased from the New England Nuclear Corp. and [2-'4C]fluorodeoxyuridine from Schwarz/ Mann. The nucleoside was converted to FdUMP with POC13 by a micromodification of the procedure of Yoshikawa et al. (12). Thymidylate synthetase was purified and crystallized as described by Galivan et al. (13). Preparation of Thymidylate Synthetase Containing Four Groups. Thymidylate synthetase (135 S_[14CjCarboxymethyl mg, 1.9 ,mol) was dissolved in 5 ml of a solution containing 6 M guanidine-HCl, 1 M Tris-HCl (pH 8.0), and 0.1 M 2-mercaptoethanol. The enzyme solution was incubated for 4 hr at room temperature under a nitrogen atmosphere and then reacted for 10 min with 8.5 mg of iodo[1-14C]acetate (27.2 .Ci/mg). After this period, 94 mg of unlabeled iodoacetate were added and the incubation was continued for another 10 min. 2-Mercaptoethanol (35 ,ul) was then added, and the resulting solution was dialyzed in the cold against four changes of 6 liters each of 5% acetic acid. Amino-acid analyses were used to determine the extent of S-carboxymethylation. Preparation of Synthetase with One S{14C]Carboxymethyl Group. Thymidylate synthetase (5 mg, 72 nmol) was passed through a Sephadex G-25 column (1 X 25 cm) with 0.05 M potassium phosphate (pH 7.0) to remove 2-mercaptoethanol. The enzyme was then treated at room temperature with iodo[ I 14C]acetate (7.2 iAmol, 3000 cpm/nmol) in 2.0 ml of 0.1 M Tris.HCI (pH 8.0) under an argon atmosphere. After 60 min it was 96% inhibited, even when assayed in the presence of excess 2-mercaptoethanol. From 0.95 to 1.1 mol of iodoacetate was incorporated per mol of enzyme, as determined by a nitrocellulose assay (2). The carboxymethylated enzyme was dialyzed against three changes of 2 liters each of 0.1 M 1848
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Bellisario et al. NH4HCO3 containing 2-mercaptoethanol for a total of 18 hr. The residual unreacted cysteinyl residues were then carboxymethylated with unlabeled iodoacetate (14). Preparation of [2-14C]FdUMP-Enzyme Ternary Complex. One hundred milligrams (1.4 imol) of thymidylate synthetase were dissolved in 5 ml of 0.1 M 2-mercaptoethanol and dialyzed for 16 hr against two 1-liter changes of 0.05 M potassium phosphate (pH 7.0) and 0.02 M 2-mercaptoethanol. The dialyzed enzyme, with a specific activity of 3.4 units/mg of protein (3), was converted to a ternary complex by incubating with 8 gmol of L-5,10-CH2H4 folate and 3.3 ,umol of [214C]FdUMP (3 X 106 cpm/gmol) at 300 for 150 min in the dark. The complexed enzyme was S-carboxymethylated with unlabeled iodoacetate as described above and dialyzed against three 4-liter changes each of 10% acetic acid and 5 mM 2mercaptoethanol, followed by lyophilization. According to the nitrocellulose filter assay (2), 2.2 mol of [2-14C]FdUMP per mol of enzyme had been incorporated, and of this, 72% (1.6 mol of FdUMP per mol of enzyme) was still bound after dialysis. Isolation of Labeled Peptides After Cyanogen Bromide Treatment. The [14C]Cm- and [2-14C]FdUMP-enzyme complexes were converted to peptides with cyanogen bromide by the procedure of Steers et al. (15). The extent of cleavage was followed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea (16) and by amino-acid analysis for methionine. The peptides were separated on an SP-Sephadex column by a modification of the procedure of Langley et al. (17) (Fig. 1). Amino Acid and Sequence Analysis. Peptides were hydrolyzed with 6 M HC1 at 110° for 22 hr in evacuated, sealed tubes. To minimize destruction of Cm-cysteine and tyrosine (18), 1 ,ul of thioglycolic acid and 20 ,l of a 5% solution of phenol were added prior to hydrolysis. Single column amino-acid analysis was performed on a model 5AH amino-acid analyzer (Jeol Co.) with Durrum DC-lA resin and pico-buffers. Manual sequencing with 10 nmol of peptide was performed by a modification (19) of the dansyl-Edman procedure (20). The coupling buffer contained 200 ,ul of trimethylamine (25% aqueous solution)-pyridine-water (0.5:4:2, vol/vol) and 5 ,l of phenylisothiocyanate. Dansyl amino acids were identified by thin-layer chromatography on polyamide sheets (5 X 5 cm) (21). Automatic peptide sequencing was performed with a Beckman model 890B sequencer using the 0.1 M Quadrol peptide program of Brauer et al. (22). The amino-acid phenylthiohydantoins were identified by a combination of thinlayer chromatography on silica gel sheets (23, 24), and isothermal gas-liquid chromatography (25). Hydrolysis of Peptides with Leucine Aminopeptidase and Carboxypeptidase A. Samples of peptide (10 nmol) were hydrolyzed for 21 hr at 370 with leucine aminopeptidase (0.2 nmol) in 0.1 ml of 0.1 M Tris-HCl (pH 8.6) containing 2.5 mM MgCl2. The reaction was terminated by the addition of 10Qul of 1 M HCI and 0.9 ml of Durrum pico-buffer A. The resultant solution was applied immediately to the amino-acid analyzer. For carboxypeptidase A treatment, 10 nmol of peptide was hydrolyzed for 24 hr at 25° with 0.2 nmol of enzyme in 0.1 ml of 0.1 M potassium phosphate (pH 8.2). The hydrolysis products were subjected to amino-acid analysis as described for leucine aminopeptidase. Limited carboxypeptidase A digestions were conducted at 250 with 0.6 nmol of peptide and 0.6 pmol of enzyme. At intervals of 15, 30, and 60 min, aliquots containing 0.2 nmol of peptide were removed and frozen immediately in liquid nitrogen. After Iyophilization, the hydrolysis products were dansylated and identified by thin-layer chromatography
1849
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FIG. 1. SP-Sephadex chromatography of cyanogen bromide peptides from (Cmj2-14CJFdUMP)2- or (["C]Cm)4-thymidylate synthetase. The cyanogen bromide peptides from 100 mg of enzyme were dissolved in 0.01 M ammonium formate (pH 2.8) containing 6 M urea and passed through an SP-Sephadex column (1.5 X 25 cm), which was then washed with 100 ml of the ammonium formate-urea solution. The column was next eluted (A to B) with a linear gradient of 0-1 M NaCl, with each reservoir containing 300 ml of the 0.01 M ammonium formate-6 M urea. On completion of the gradient, the labeled CN2 peptide (shaded area) was eluted from the column with 0.5 M NH4OH-6 M urea (C). Urea was removed from the pooled peptide fractions by dialysis against 30% acetic acid in Spectropor 3500 dialysis tubing (Spectrum Medical Industries).
on polyamide plates. Appropriate enzyme blanks were included for each proteolytic reaction. Isolation of Radioactive Chymotryptic Peptides from [14CJCm-CN2. To approximately 90 nmol of (['4C]Cm)2-CN2 (9 X .105 cpm) was added a 10-fold excess of unlabeled peptide. The combined peptides (1.0 ,umol; 9 X 105 cpm) were suspended in 3 ml of 1% NH4HCO3 containing 0.1 mg of chymotrypsin. After 6 hr at 370 the suspension cleared, and 0.1 mg more of enzyme was added. Hydrolysis was continued for a total of 16 hr at 370, at which time the solution was lyophilized. The residue was dissolved in 1 ml of 10% acetic acid and fractionated on a column of BioGel P-2 (1.5 X 97 cm), with further purification of the radioactive peptides by two-dimensional ascending chromatography on Whatman 3MM paper. Solvent 1 was n-butanol-acetic acid-water (4:1:5, vol/vol); solvent 2, n-butanol-20% pyridine acetate (pH 5.4)-water (1:1:1, vol/vol). The 14C-labeled peptides, detected by radioautography with Kodak RP/R2 x-ray film, were eluted from the paper with 20% pyridine acetate (pH 5.4). To assess the purity of the peptides, analytic two-dimensional mapping on cellulose thin-layer plates (20 X 20 cm) was carried out as described by Schachner and Zillig (26). Nonradioactive peptides were detected with 0.2% ninhydrin in acetone.
RESULTS Cyanogen bromide cleavage of thymidylate synthetase. In general agreement with the findings of Loeble and Dunlap (27), four distinct bands and one or two less visible bands were obtained when the cyanogen bromide-treated carboxymethylated enzyme was electrophoresed in polyacrylamide gel containing urea and sodium- dodecyl sulfate. The largest band (CN1) had a molecular weight of about 13,000; the smallest
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Proc. Nati. Acad. Sci. USA 73 (1976)
Table 1. Comparison of amino-acid content of peptide derivatives from thymidylate synthetase
(Cm, FdUMP)2Synthetase (residues/70,000 g)
Amino acid
(Cm)4-Synthetase
Cm-Cys Asp Thr Ser Glu Pro Gly Ala Val Met Ile
3.54 71.2 31.4 23.2 56.6 30.2 37.1 43.4 26.6 11.9 25.9 68.7 27.8 38.0 35.5 35.7 23.5
Leu Tyr Phe Lys His Arg
(Cm, FdUMP)CN2 (residues/10,000 g)
(Cm)2-CN2
1.97 71.8 34.2 21.2 56.0 31.4 36.7 43.4 26.2 12.0 25.8 67.5 27.4 38.4 36.2 37.0 24.6
1.26 10.9 4.01 3.59 9.20 5.98 4.85 6.05 3.52
C2A
C2B (residues)*
0.87 10.8
1.0
1.09
4.09
1.04
0.85
3.49 9.44 6.04 5.16 6.31 4.08
3.60 1.98 2.44 0.91 2.18
0.97
1.92
2.26 1.03
0.48t
0.49t 3.64 12.6 4.09 5.71 2.89 5.68 2.70
4.10 12.2 4.09 5.49 2.85 5.66 2.78
0.97 1.90
0.97
* Mol/mol of peptide. t Based on recovered homoserine lactone.
band was about 2000, but because bands of this size and smaller tended to elute from the gel on destaining it was difficult to determine the number of such bands. The sum of the calibrated molecular weights of the peptide bands approximated 35,000, in good agreement with that reported for a subunit of the L. casei thymidylate synthetase (3). When the native enzyme was labeled with radioactive FdUMP, iodoacetate, or N-ethylmaleimide and then S-carboxymethylated under denaturing conditions with unlabeled iodoacetate, only the second band (CN2) was found to be radioactive. Since the enzyme is composed of two identical subunits (27), CN2 with a molecular weight of 10,000 appears to account for the two cysteines in each subunit (Table 1). It is of interest to note that carboxymethylation of the FdUMP-tre4ted enzyme yielded only two of the four Cm-cysteinyl residues usually obtained with untreated enzyme. Chymotryptic peptides from CN2 Initially, ([14C]Cm)2-CN2 was isolated by pooling selected fractions after BioGel P-10 column (1.5 X 96 cm) chromatography. This peptide was then digested with chymotrypsin; a thin-layer, two-dimensional chromatogram is presented in Fig. 2A. On BioGel P-2 chromatography of the digest, two radioactive peaks were eluted, Cm-C2A and Cm-C2B. The smaller of the two, Cm-C2B, was purified to homogeneity by two-dimensional paper chromatography (Materials and Methods) and migrated to the shaded region designated by the arrow in Fig. 2A. Amino-acid analysis of Cm-C2B after acid hydrolysis (Table 1) or leucine aminopeptidaset digestion yielded similar results, with the most significant finding being the presence of one Cm-cysteinyl residue per mol of peptide. This value represents about half that in (Cm)2-CN2. The isolated labeled C2B from ([14C]Cm)2-CN2 was analyzed by a manual dansyl-Edman procedure (18) to yield the sequence: Ala-Leu-Pro-Pro-(Cm-Cys)-His-Thr-Leu-Tyr Verification of the sequence at the carboxyl-terminal end was obtained with carboxypeptidase A; tyrosine and leucine t
Normally leucine aminopeptidase does not hydrolyze proline bonds, but this preparation was apparently contaminated with prolidase.
were released quantitatively but threonine and histidine were hydrolyzed to a lesser extent (30% and 10%, respectively). Recent studies by us, where one mol of iodo[l-'4C]acetate was fixed to one mol of enzyme to completely inactivate it, followed by unlabeled iodoacetate to carboxymethylate the remaining sulfhydryl groups, yielded a "4C-labeled chymotryptic peptide from (Cm)2-CN2 with half the specific activity of the same peptide from synthetase that had been completely S-carboxymethylated with iodo[l-14C]acetate. The radioactive peptide migrated to the same region as that occupied by Cm-C2B (Fig. 2A, arrow). The nature of the proposed reaction is indicated below: two CN2 peptides are presented to represent the two identical subunits of the synthetase, with only one of the native enzyme's four sulfhydryl groups being labeled. Cm Cm Ala fiCN2-
1
_I
["4CJCm
Cm Chymotrypsin
Cm
Cm
Ala-C2B--Tyr Ala-I-C2B-Tyr [14CJCm
+ peptides +
I
-C2A
I- -C2A
-
t Cm
After chymotrypsin treatment of (Cm,[2-"4C]FdUMP)CN2, two-dimensional chromatographic analysis indicated that the ninhydrin spot corresponding to Cm-C2B was missing (Fig. 2B, arrow) and that the radioactivity was located in a region other than that shown in Fig. 2A. This finding strongly suggests that FdUMP was present in place of one of the two Cm-residues of (Cm)2-CN2, most probably that in Cm-C2B, and is supported by the amino-acid data in Table 1 and the sequence analyses below. Isolation and sequence analysis of FdUMP- and Cm-. cysteine-containing CN2 To facilitate the isolation of the cyanogen bromide peptides, we used SP-Sephadex column chromatography described in
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Bellisario et al.
K
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1851
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FIG. 2. Two-dimensional thin-layer chromatography of a chymotryptic digest of (A) ([14C]Cm)2-CN2 and (B) (Cm,[2-14C]FdUMP)-CN2. The arrows designate the locations of the Cm-cysteine-containing peptide, C2B. The stippled regions represent those of relatively intense radioactivity and the dotted regions, those of less intensity. Solvents 1 and 2 are described in Materials and Methods.
the legend of Fig. 1. Under these conditions, both the ([14C]Cm)2- and the (Cm,[2-14C]FdUMP)-CN2 peptides were the most tightly bound, but could be released with 0.5 M NH4OH-6 M urea after the gradient elution procedure. As indicated in Table 1, the two peptides are almost identical in amino-acid composition, except for the number of Cm-cysteinyl residues, which in the case of the (Cm,FdUMP)-CN2 was always less than 1. What happens to the second cysteinyl residue on acid hydrolysis of (Cm,FdUMP)-CN2 is not known, but this fact, plus others to be discussed, indicates that the FdUMP-cysteine linkage does not yield cysteine on acid hydrolysis. Although the Cm-Cys value in (Cm)2-CN2 is closer to 1 than to 2 in this particular preparation, others have yielded values closer to 1.5. The low values for Cm-Cys are not surprising in view of the instability of this residue. More representative data, however, were obtained with the isolated chymotryptic peptides, Cm-C2A and Cm-C2B, as indicated by the amino-acid analyses in Table 1. Sequence analysis of Cm- and FdUMP-labeled CN2 Because the amino-acid compositions of (Cm)2-CN2 and (Cm,FdUMP)-CN2 were so similar, an effort was made to determine the location of the chymotryptic peptide C2B, in the 90-100 amino acids of CN2. Preliminary dansyl-Edman analysis of CN2 revealed that the nonapeptide, C2B, might be located on the amino-terminal end because a sequence of Ala-Leu-Pro was obtained. This fortunate coincidence was confirmed by automatic sequence analysis of the first 13 residues from each of the two peptides: (Cm,FdUMP)-CN2: Ala-Leu-Pro-Pro-
[X]-His-Thr-Leu-Tyr-Gln-Phe-Tyr-Val(Cm)2-CN2: Ala-Leu-Pro-Pro-
[Cm-Cys]-His-Thr-Leu-Tyr-Gln-Phe-Tyr-ValAlthough the association of radioactivity with the fifth residue of (Cm)2-CN2 confirmed the presence of cysteine at this position, comparable results were not obtained with (Cm,[214C]FdUMP)-CN2. In this case, radioactivity was obtained in several residues, particularly in the early ones, indicating that the acid conditions associated with cleavage were effecting a slow release of FdUMP. DISCUSSION Although most investigators generally agree on the nature of the ternary complex resulting from the interaction of thymi-
dylate synthetase with 5,10-CH2H4 folate and FdUMP, some question has arisen concerning the nucleophile that initiates complex formation. Danenberg et al. have proposed (5), because of the decrease in absorbance at 270 nm, that a cysteine sulfhydryl saturates the 5,6-double bond of the pyrimidine ring of FdUMP. This in turn is believed to promote a methylene bridge structure between 5,10-CH2H4 folate and the 5-position of the enzyme-FdUMP complex (5, 28), as originally proposed by Friedkin and Kornberg (29). The studies of Santi and coworkers, however, although in general agreement with the ternary complex thesis, are not in harmony with respect to the nucleophile. Thus, their studies with N-ethylmaleimide (30), in which 4 mol were found to react with the FdUMP-enzyme complex, and their isolation of an FdUMP-peptide (10) containing [Thr, His, Ala, Leu, (Pro)2], have led them to propose threonine or histidine as the nucleophile. This peptide is probably a smaller version of C2B isolated by us. Studies by others with sulfhydryl reagents, however, have shown that the synthetase can be completely inhibited by pchloromercuribenzoate (3), N-ethylmaleimide (3), iodoacetamide (6), and showdomycin (31), and can be protected by the substrate, dUMP (5). It appears that reaction of only one of the enzyme's four sulfhydryl groups is sufficient to effect complete inhibition (3, 6). Similar to the findings of Dunlap et al. (3) with p-chloromercuribenzoate, we obtained identical results with N-ethylmaleimide and other compounds to be reported. In addition, by amino-acid analyses of the products of the reaction of N-ethylmaleimide with the FdUMP-enzyme complex under conditions similar to those described by McHenry and Santi (30), we have found that sufficient amino acids other than cysteine react to account for 4 mol of N-ethylmaleimide per mol of FdUMP-enzyme complex. When the number of Nsuccinyl cysteinyl residues from FdUMP-treated synthetase was compared with that from untreated enzyme, approximately two fewer residues were found in the treated enzyme, indicating that FdUMP had protected two of the enzyme's four sulfhydryl groups from N-ethylmaleimide. Similar results, but with less nonspecific reaction, were obtained on iodoacetate treatment of the FdUMP-enzyme complex under denaturing conditions. Recent studies (11) with 3SS-labeled enzyme are also in agreement with the presence of cysteine at the FdUMP reactive site.
The reason for the apparent absence of cysteine in the acid-hydrolyzed FdUMP-peptide obtained by Sommer and Santi (10) and by ourselves is not apparent at present, although
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Biochemistry: Bellisario et al.
there is no reason to assume that an FdUMP-cysteine adduct should yield cysteine on acid hydrolysis. Alternatively, it should not be ruled out that the peptide isolated by them does not contain cysteine, but is coincidently similar in amino-acid composition to the one isolated by us. It is difficult, however, to deny an association of FdUMP with Cys5 of CN2, based on the sequence analyses presented for (Cm,FdUMP)-CN2 and (Cm)2-CN2 and the amino-acid data of Table 1. These data are consistent with the less drastic conditions of leucine aminopeptidase hydrolysis, where the other proposed nucleophiles (10), threonine and histidine, could be accounted for in both peptides. The above results indicate that FdUMP and cysteine are linked in some, as yet, undefined manner. It should be emphasized that these findings do not imply that cysteine is the nucleophile that initiates catalysis at the active site or that dUMP and FdUMP react identically at this site. Although logic dictates that this may be the case because of the structural similarities of dUMP and FdUMP, other factors must also be considered. Thus, although FdUMP reacts initially as a competitive inhibitor of dUMP, the reaction does become noncompetitive with time (32-34) and FdUMP eventually becomes covalently bound to two sites on the enzyme. In contrast, recent circular dichroism (6) and binding studies (35) with dUMP indicate that dUMP binds primarily to a single site on the synthetase, in agreement with the inhibition experiments with sulfhydryl reagents. Whether dUMP and FdUMP react initially at the same site, and FdUMP becomes covalently bound to that site or is transferred subsequently to another site where it becomes irreversibly bound, remains to be determined. We thank Don U. Guarino for his excellent technical assistance and Dr. B. Pollara of the Kidney Disease Institute for permitting us the use of his sequenator. This research was supported in part by Public Health Service Grant GM-20371 from the National Institute of General Medical Sciences, PHS/DHEW. 1. Langenbach, R. J., Danenberg, P. V. & Heidelberger, C. (1972) Biochem. Biophys. Res. Commun. 48,1565-1571. 2. Santi, D. V. & McHenry, C. S. (1972) Proc. Natl. Acad. Sci. USA 69, 1855-1875. 3. Dunlap, R. B., Harding, N. G. L., & Huennekens, F. M. (1971) Biochemistry 10, 88-97. 4. Leary, R. P. & Kisliuk, R. L. (1971) Prep. Biochem. 1, 47-54. 5. Danenberg, P. V., Langenbach, R. J. & Heidelberger, C. (1974)
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