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Studies on identifying the folylpolyglutamate binding sites of Lactobacillus casei thymidylate synthetase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 216, No. 2, July, pp. 551-558, 1982
Studies on Identifying Lactobacillus GLADYS
F. MALEY,*
the Folylpolyglutamate Binding Sites of casei Thymidylate Synthetase’
FRANK
MALEY,*T~
AND
CHARLES
M. BAUGHT
*Division of Laboratories ad Research, New York State Department of Health, Albang, New York 12201; and the tDepartment of Biochemistry, College of Medicine, University of South Alabama, Mobile, Alabama 36688 Received
January
22, 1982
Pteroylheptaglutamate labeled with 14C in its proximal glutamate was activated with a water-soluble carbodiimide and reacted with Lactobacillus casei thymidylate synthetase in the presence and absence of dUMP. The latter nucleotide appeared to enhance the specificity of the reaction as the quantity of folylpolyglutamate fixed plateaued at 1.5 mol/mol of enzyme, but continued to increase above 2.0 in the absence of dUMP. The location of the covalently bound pteroylheptaglutamate was determined by first cleaving the labeled protein with cyanogen bromide and establishing that 80% of the radioactivity was present in the second of the five cyanogen bromide peptides of thymidylate synthetase. This peptide was then digested with chymotrypsin and the resulting peptides were purified by high-performance liquid chromatography. Two labeled peptides were isolated, with one being separated further into two pure peptides on gel exclusion chromatography. The three peptides represented residues 48-54, 52-61, and 55-61 of the synthetase, with the pteroylheptaglutamate fixed covalently to lysines 50 or 51 and 58. From the results presented, it would appear that each of the enzyme subunits is labeled, with the folylpolyglutamate being fixed to lysine 58 of one subunit and 50, 51 of the other.
Many, if not most of the antifolate drugs were designed originally to inhibit dihydrofolate reductase. However, it has been found that these compounds can also impair thymidylate synthetase (l-4), particularly in the presence of compounds which potentiate the binding of the folates such as the enzyme’s nucleotide substrate dUMP3 and its analogs (5, 6). Thus
the binding of PteGlu, H2PteGlu, 5,10CH2H4PteGlu, and folate analogs such as MTX is not detectable in the absence of dUMP or FdUMP but are bound in the presence of either nucleotide (5). Even in the case of the predominant forms of folate found in nature, the folylpolyglutamates, which bind to the synthetase in the absence of dUMP and related nucleotides, binding is greatly enhanced in the presence of the latter compounds (7). In view of these results it was not surprising to
i This investigation was supported in part by Public Health Research Grants GM26387 and GM26645 from the National Institutes of General Medical Sciences, PHS/DHHS. ‘Author to whom correspondence should be addressed. a Abbreviations used: dUMP, 2’-deoxyuridylate; FdUMP, 5-fluoro-2’-deoxyuridylate; PteGlu, pteroylglutamate (folate); HaPteGlu, dihydrofolate; loCH&PteGlu, lo-methyltetrahydrofolate; lo-CHOH4PteGlu, 10 - formyltetrahydrofolate; 5,10-CHa -
H,PteGlu,5,10-methylenetetrahydrofolate; PteGlua, pteroyltriglutamate; HaPteGlu,, dihydropteroylpentaglutamate; PteGlu7, pteroylheptaglutamate; MTX, methotrexate; CNBr, cyanogen bromide; TLCK, tosyllysine chloromethyl ketone; HPLC, high-performance liquid chromatography; TCA, trichloroacetic acid; PTH, phenylthiohydantoin. 551
0003-9861/82/080551-08$62.00/O Copyright All righta
8 1982 by Academic Press, Inc. of reproduction in any form reserved.
552
MALEY.
MALEY.
find that the polyglutamate derivatives of 5,10-CHzH4PteGlu are considerably better substrates than 5,10-CHzH4PteGlu (8), a finding supported by the recent observation that the tightness of ternary deadend complexes of FdUMP-synthetase and the polyglutamates of 5,10-CHzH4PteGlu varies directly with the number of glutamates, up to five residues (9). Similarly, in those cases where protection of the enzyme against proteolytic degradation was measured the resistance to proteolysis was found to increase with the number of glutamates on folate (lo), an effect which implicates electrostatic interactions between the carboxyls of glutamate with a positively charged region of the enzyme. To complement our studies on identifying the nucleotide binding sites within the primary structure of thymidylate synthetase, which have revealed where FdUMP and most probably dUMP bind (ll), we have undertaken in this study to locate the region responsible for enhancing folylpolyglutamate binding. EXPERIMENTAL
PROCEDURE
Materials L cusei thymidylate synthetase was obtained from the New England Enzyme Center, Boston, Massachusetts, as a dialyzed ammonium sulfate fraction and purified to homogeneity as described previously (6). Folylpolyglutamates, including Pte[U-14C]GluGlus, which had a specific activity of 1 &i/pmol (1600 cpm/ nmol) were synthesized as described previously (12). Sephadex G-100 (loo200 mesh) and Sephadex G-25 (medium) were purchased from Pharmacia and BioGel filtration media were obtained from Bio-Rad. 1-Ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride was purchased from Pierce Chemical Company. TLCK-a-chymotrypsin was purchased from Worthington and TPCK-treated trypsin was kindly provided by Dr. T. H. Plummer, Jr. All other chemicals were of reagent grade or HPLC grade. Methods Fixation of Pte[U-‘4CJGluGlu+ Crystalline thymidylate synthetase was dissolved
AND BAUGH
in a minimum volume of 50 mM potassium phosphate, pH 7.1, 20 mM 2-mercaptoethanol and dialyzed against two changes of 2 liters each of the same buffer. The fully activated enzyme had a specific activity of 3.6 pmol/mg when assayed in the absence of Mgz+ and 5.3 pmol/mg when Mgz+ was present. The assay employed was a slight modification of the spectrophotometric procedure of Wahba and Friedkin (13). In each case the assay buffer was at pH 7.0. The activated enzyme was then passed over a column of Sephadex G-25, equilibrated with 50 mM potassium phosphate, pH 7.1, to remove the 2-mercaptoethanol from the enzyme solution. The resulting enzyme solution, 30 mg (0.43 pmol) in 3.0 ml, was mixed with 0.1 ml of 0.1 M dUMP. In a separate tube, 0.5 ml of 6 mM Pte[Ur4C]GluGluG (1600 cpm/nmol) was added to 0.5 ml of freshly prepared 10 mM l-ethyl - 3 - (dimethylaminopropyl)carbodiimide hydrochloride in 50 mM NaHC03, pH ‘7, similar to that described for the activation of methotrexate by Chu and Whiteley (14). The enzyme-substrate solution was then mixed with the labeled polyglutamatecarbodiimide adduct and allowed to react at room temperature in the dark. Small aliquots of 20 ~1 were removed at 30 and 60 min and assayed for binding with a nitrocellulose filter (5). The entire reaction was terminated at 60 min by the addition of 0.5 ml cold 50% TCA. After cooling, the resulting precipitate was centrifuged and the residue washed several times with 6 ml cold 5% TCA. An average yield of the polyglutamate bound adduct was 800 nmol of thymidylate synthetase monomer with 600 nmol of bound Pte[U-14C]GluGlus. All measurements of radioactivity were determined with a Packard Model 3320 Tri-Carb liquid scintillation counter using Aquasol as the counting medium. Chemical and enzptatic cleavage pm cedures. Thymidylate synthetase (29 mg) labeled with Pte[U-14C]GluGlus was cleaved with cyanogen bromide and the peptides were separated by extraction and column chromatography procedures as described previously (15). Labeled CNBr2 was subjected to chymotryptic hydrolysis at pH
FOLYLPOLYGLUTAMATE
AND L Casei THYMIDYLATE
8.2 to generate labeled peptides for compositional analysis. Reversed-phase high-perfiwmance liquid chromatography. Cyanogen bromide and chymotryptic peptides were purified by reversed-phase high-performance liquid chromatography with a Beckman Altex instrument containing a Waters PBondapak Cl8 column (0.4 X 25 cm). A linear gradient of 0.1% phosphoric acid to 90% acetonitrile/O.Ol% phosphoric acid was employed to effect the desired separations. In some cases, peptides were purified further by rechromatography on HPLC, followed by Bio-Gel-P2 column chromatography (1.5 X 85 cm) with 20 mM ammonium bicarbonate as the eluant. Amino acid analysis. Labeled protein and peptides were hydrolyzed for 24 h at 110°C in evacuated, sealed tubes containing 1.0 ml of constant boiling HCl. Single column amino acid analysis was performed on a Beckman Model 119CL amino acid analyzer. Edman degradaticms. Sequence determinations were performed with a Beckman Model 890 B automatic sequencer. Peptides were sequenced using N-alkyl-Ndimethylamine as the buffer (16). The released thiazolinone derivatives were identified after conversion to their respective PTH-amino acids by thin-layer chromatography and HPLC (15, 16). RESULTS AND DISCUSSION
Nature
of the Folate Binding
Site
When the effect of various folate analogs on the kinetics of 5,10-CHzH4PteGlu utilization is determined, it would appear that more than one site is involved in the binding of these compounds. Thus, as first shown with the chick embryo synthetase HzPteGlu is a noncompetitive inhibitor (17), which was shown subsequently to be the case for the human leukemic enzyme (2). In the latter instance MTX, PteGlus, and HzPteGlus were found also to be noncompetitive inhibitors of 5,10-CHzH4PteGlu while PteGlu, lo-CHsHIPteGlu, and lo-CHO-HIPteGlu appeared to be competitive inhibitors. On measuring the kinetics of interaction of MTX with the L casei synthetase, it too behaved as a non-
SYNTHETASE
mM-’
553
FHI
FIG. 1. Nature of the inhibition of thymidylate synthetase promoted by PteGlu7 and methotrexate. The reaction mixtures at 30°C contained 5 mM ascorbate, 50 mg 2-mercaptoethanol, (dl)t-H,PteGlu (FH,) as the variable substrate, 3.2 mM formaldehyde, 0.5 mM dUMP, 50 mM MgC12, and 50 mM Tris-HCl at pH 7.0. PteGlu7 and MTX were present at the indicated levels and the reactions were initiated by addition of enzyme. The spectrophotometric procedure of Wahba and Friedkin (13) was used to determine the initial velocity of each reaction.
competitive inhibitor as shown in Fig. 1. In contrast, it is clearly seen that PteGlu7 acts in a competitive manner, which appears to contrast with results obtained for the polyglutamates and the human synthetase (2). However, it is possible that the latter data are a consequence of only single time points being used to determine initial velocities, or possibly to an inherent difference in binding properties of the two enzymes. In any event it appears from the results in Fig. 1 that PteGlu, competes with 5,10-CHzH4PteGlu for its site on the L. casei synthetase, signifying that the pteroyl moieties overlap in their binding. Additional evidence favoring this thesis has come from nitrocellulose binding (5) in which the enzyme’s folate site was protected by first fixing 5,10-CHzH4PteGlu to the enzyme as part of the FdUMP ternary complex and then measuring the ability of [14C]PteGlu7 to bind. It was found that only about 20% of the PteGlu7 fixed to the complexed enzyme relative to the free enzyme. That which was bound to the ternary complex probably represents nonspecific binding. The greatly enhanced binding of the pteroyl polyglutamates relative to the monoglutamate is probably due to the greater opportunity for electrostatic interaction between the carboxyl anions of the polyglutamate residues and the lysyl
554
MALEY,
MALEY,
cations of a specific region of the protein. To identify this region, the carboxyls of the ultimate glutamate of PteGlu7 were activated, possibly to an (Y, y-anhydride, through the use of a water-soluble carbodiimide. The resulting anhydride could then covalently link to the c-amino groups of lysines in its proximity to facilitate the location of these residues within the primary sequence of the protein. Since dUMP enhances the binding of folate and its derivatives (6), including the folylpolyglutamates (7), probably by increasing their binding specificity, this nucleotide was included in the reaction solutions. If dUMP was not incuded, the binding of compounds such as PteGlu7 increased progressively with concentration to above 2 mol/mol of enzyme. However, in the presence of dUMP its binding plateaued at about 1.4 mol/mol of enzyme, a result which implies that the binding of PteGlu, is less random in the nucleotide’s presence (Fig. 2).
Identifying the PteGlu, Binding Site Once fixed to the enzyme, the location of PteGlu, was established by employing a procedure similar to that described for the FdUMP site (18). The labeled protein was cleaved with cyanogen bromide to its five peptides, which were purified by a combination of solvent extraction and BioGel column chromatography. From the
PteGlu,
ADDED
FIG. 2. Fixation of Pte[U-‘“C]GluGlus to thymidylate synthetase in the presence and absence of dUMP. The folylpolyglutamate was activated by a water-soluble carbodiimide and the degree of irreversible fixation was measured by a nitrocellulose filter assay (see Experimental Procedures for details).
AND
BAUGH TABLE
I
DISTRIBUTION OF RADIOACTIVITY IN THE CNBr PEPTIDES’ FROM L casei Pte[14C]GluGlus THYMIDYL.ATE SYNTHETASE CNBr peptide 1 2 3 4 5
Radioactivity (cpm/nmol)
Relative amount (%)
2 1225 49 225 4
0.1 81.4 3.2 15.0 0.3
a The CNBr peptides were isolated as described in (15). The peptides were further purified by reversedphase HPLC as described under Experimental Procedures.
distribution of 14C in the various CNBr peptides (Table I), it is obvious that most of the label (about 80%) is in CNBr2 as opposed to only 15% in CNBr4, the peptide to which FdUMP binds (15, 18, 19). That the peptide containing the folate was indeed CNBr2 was confirmed by amino acid analysis (Table II), which reveals ‘7 mol TABLE
II
AMINO ACID COMPOSITIONSOF CNBr2 AND PtdU-‘“C]GluGlus-CNBr2 Amino acid
Labeled CNBr2’
CNBrZb
Asp Thr Ser Glu” Pro GUY Ala Val Ile Leu 5r Phe LYS His Aw
a.7 3.9 3.8
9 4 4
15.9
9
3.6 5.3 2.6 2.6 2.7 9.0 1.4 7.8 7.2 5.5 3.8
4 5 3 3 3 9 2 8 7 6 4
a The values were obtained from 24-h acid hydrolysates and represent molar ratios relative to leucine. The specific radioactivity of purified labeled CNBr2 was 1266 cpm/nmol. b Composition of CNBr2 as determined previously (19). c Italics are used to emphasize increase in Glu residues from 9 to 15.9.
FOLYLPOLYGLUTAMATE
AND
L Casei THYMIDYLATE
TABLE
AMINO ACID COMWSITION Amino acid Thr Ser Glu Pro GUY Val Ile Leu Phe LYS
OF LABELED
Cl’ (nmol) 2.93 (1.51)
2
15.21 (7.34) 2.30 (1.19)
7
2.22 (1.14)
1
2.13 (1.10) 3.99 (2.01)
Position in CNBr2 cpm/nmol”
III
CHYMOTRYPTIC PEPTIDES FROM Pte[*%]GluGly Theor
1
1 2
C2 (nmol)
Theory
3.15 (1.26) 16.49 (6.60) 2.27 (0.91) 3.09 (1.24)
2.51 (1.00) 1.89 (0.76) 3.69 (1.48) 2.80 (1.12) 3.08 (1.24)
48-54 1110
555
SYNTHETASE
LABELED CNBr2
C3 (nmol)
Theory
1.59 (0.97) 11.95 (7.29)
1 8
1.77 (1.04)
I
1.43 (0.90)
1
2.87 (1.75)
2
2.13 (1.30)
I
52-61 1170
55-61
1130
’ Cl, C2, and C3 represent the three “C-labeled chymotryptic peptides in their sequential location in CNBr2. *The figures in parentheses are the estimated number of residues of each amino acid per peptide. For each peptide the number of nanomoles for all amino acids except Glu were added, and divided by the theoretical number of all amino acids except Glu to give an estimate of the number of nanomoles of the peptides (1.94 nmol for Cl, 2.50 nmol for C2, and 1.64 nmol for C3). The number of nanomoles of each amino acid was then divided by this value to give the estimated number of amino acid residues per peptide. ’ Specific radioactivities of the isolated peptides.
more of glutamate than expected for pure CNBr2, but in accord with that expected for a peptide containing PteGlu,. To further define the region occupied by PteGlq within CNBr2 this peptide was digested with chymotrypsin and the resulting peptide mixture separated by HPLC. From the elution profile presented in Fig. 3, it was apparent that most of the label was in two closely associated peaks. Further purification of these peptides on Bio-Gel-P2 resulted in the resolution of the slower eluting peptide into two peptides (C2 and C3). Amino acid analysis of the three purified peptides indicated that each contained 7 to 8 mol of glutamate/ mol of peptide (Table III). The fact that the number of glutamate residues in the isolated peptides differ by about one from theory is probably due to the use of an average value in calculating the number
A200
MINUTES
FIG. 3. Separation of chymotryptic peptides of CNBr2 labeled with Pt@-“CJGluGl~ by HPLC. The shaded areas represent regions where radioactivity was detected. The elution of the peptides was measured at both 236 and 210 nm. For additional details see Experimental Procedures.
MALEY,
556
MALEY.
of residues (Table III). Although peptides C2 and C3 each contained one lysine to which one equivalent of PteGlu7 was covalently linked Cl contained two tandem lysines, with only one apparently linked to PteGlu,, based on the molar ratio of glutamate to lysine. Sequence and amino acid analysis of each peptide revealed them to possess the indicated order of amino acids shown in Table IV, which reveals that chymotrypsin affected not only the anticipated cleavage between residues 54 and 55 (Phe-Gly) but also one that was unexpected, that between residues 51 and 52 (Lys-Val). Of interest are the specific radioactivities of the three peptides (Table III), which in each case reflects a 25% reduction in the specific activity of the starting material, PtflJ4C]GluGlus. This dilution was anticipated from the results in Fig. 2, which revealed that only about ‘75% of the enzyme had been labeled, a result which was confirmed by the finding that CNBr2 possessed the same specific radioactivity as the isolated chymotryptic peptides (cf. Tables I and III). Since the enzyme is composed of two identical subunits and the chymotryptic peptides were derived from both, it was not possible to distinguish between the labeling of one subunit by 1.5 mol of PteGlu, or the asymmetric labeling of each enzyme subunit by 0.75 mol of PteGlu,. Thus a situation can be envisaged TABLE PROBABLE
PEPTIDE
IV
SEQUENCES BY [“CjPteGlu,”
IN CNBr2
LABELED
(1) Thr-Thr-Lys-Lys-Val-Pro-Phe (2) Val-Pro-Phe-gy-Leu-Ile-Lys-Ser-C%Leu (3)
60 Gly-Leu-Ile-Lys-Ser-Glu-Leu
‘Peptide (1) represents the faster moving of the two labeled peaks in Fig. 3, while peptides (2) and (3) are from theslower moving peak, which was fractionated into these peptides on a Bio-Gel P2 column. Amino acid analysis (Cl, C2, and C3 in Table III) and partial sequencing established their location within the primary sequence of the synthetase as described under Experimental Procedures.
AND BAUGH TABLE
V
AMINO ACID COMPOSITION OF LIMITED TRYPSIN PEPTIDE CONTAINING Pte[U-“C]GluGlus Labeled peptide Amino acid
nmol
Residues”
Theoryb
Asp Thr Ser Glu Pro GUY Ala Val Ile Leu 5r Phe Tw LYS His Aw
8.66 7.95 5.39 21.66 6.99 8.61 0.35 3.10 5.11 19.3 0.00 10.57 ND 10.47 3.04 2.47
3.4 3.2 2.1 8.7 2.7 3.3 1.2 2.0 7.7 4.2 4.2 1.2 1.0
3 3 2 8” 2 3 1 2 7 4 1 4 1 1
o The values were obtained from 24-h acid hydrolysates and represent molar ratios relative to arginine. bThe composition of this limited trypsin peptide (residues 37-72) is based on a similar peptide isolated previously from CNBr2 (19). ‘Based on the one glutamate associated with this peptide plus the seven from PteGlu,.
where PteGlu, is fixed to residues (50, 51)4 and 58 of the same subunit resulting in a peptide specific radioactivity which is about 1.5 that of the initial specific radioactivity of PteGlu,, or one in which each subunit is labeled, but assymetrically (residues 50, 51 of one subunit and residue 58 of the other). In the latter case the specific radioactivity of the enzyme subunit, as reflected in CNBr2, would be 0.75 times the specific radioactivity of PteGlu7. If residues 48-61 could have been isolated as an intact peptide its specific radioactivity would have provided a solution to this problem, but since chymotrypsin had affected cleavages between residues 51 and 52 as well as between 54 and 55 it was not possible to obtain this peptide intact; nor ’ Since it is not possible at this time to distinguish which of the two lysines was labeled, both are listed.
FOLYLPOLYGLUTAMATE
AND
L Casei THYMIDYLATE
SYNTHETASE
557
SER-LYS-GLY-PHE-PRO-LEU cl
~EIJ LEU-TRP-PHE-LEU-H~~-GLY-ASP-~HR-A~PN-ILEILE-TR~-A~~-GL~-TR~-ALA-PHE-GL"-L~~-~~~-V~L-L~~-SER-A~~-GL"-TYR-~,~-G~Y-PRO-
A !f-
MET-THR-A~~-PHE-GL~-HIS-ARG-SER-GL~-~~~-A~~-P~O-G~"-P"E-A~~-AL*-V~L-TYR-~,~-
t f?-
GLU-MET-ALA-LVS-PHE-ASP-ASP-ARG-VAL-
5%I~s-Asp-ASP-ALA-PHE-ALA-ALA-LYS-TYR-
t f-
ASP-LEU-GLV-LEU-VAL-TYR-GLY-SER-GLN-
+%-ARG-ALA-TRP-His-THR-SWLYS-GLY-Asp-
+ !i-
ILE-ASP-GLN-LEU-GLY-ASP-VAL-ILE-GLU-
t So-IKE-LYS-THR-His-PRO-TYR-SER-ARG-ARG-~-
FIG. 4. The primary sequence of L casti thymidylate synthetase designating the active site region for FdUMP (area encompassing cysteine-198) and the region labeled by PteGlu, (area encompassing lysine 50, 51 and lysine 58).
was it possible to establish whether residue 50 was labeled in preference to 51 as a result of the inability of trypsin to cleave the Lys-Lys bond. In an effort to resolve the problem of which subunit was labeled by PteGluT, this compound was fixed to the intact enzyme as described in the methods, and the complex was subjected to citraconylation and trypsinization. The limited tryptic peptides were partially separated on a BioGel-P10 column and from those fractions containing 14C, a peptide was purified to homogeneity by HPLC. Amino acid analysis of this limited tryptic peptide (Table V) revealed that it most probably corresponded to residues 37-72 of the known sequence of the L. cusei synthetase (11). The specific radioactivity bf this peptide was 1400 cpm/nmol, which was as expected somewhat less than the specific radioactivity of the initial PteGlu,, indicating that only one PteGlu, had been fixed to each subunit. This peptide, which is
basically a mixture of residues 37-72 from each subunit, was then treated with chymotrypsin and the resulting peptides were isolated by HPLC as described in the methods and Fig. 3. Amino acid analysis and radioactivity determinations revealed that two of the chymotryptic peptides, encompassing residues 48-54 and 55-61, possessed the same specific radioactivity as the starting limited tryptic peptide, suggesting that the initial fixation of PteGlu, had been asymmetric. CONCLUSIONS
From the known sequence of CNBr2 (15), it is clear that the three chymotryptic peptides are derived from residues 48-61 of thymidylate synthetase as a result of cleavages at Lys-51, Phe-54, and Leu-61. The location of this region relative to that of the previously described FdUMP containing chymotryptic peptide is presented in Fig. 4.
558
MALEY,
MALEY,
Since the region to which the folate substrate or analog is fixed is obviously dependent on the length of the glutamate chain, the binding regions of other pteroylmono- and polyglutamates and their derivatives will possibly provide an important parameter for establishing their locus of action within the topography of the synthetase. Preliminary studies have shown that MTX, which is a noncompetitive inhibitor of 5,10-CHzH4PteGlu, in contrast to PteGlu7, binds to a different region of CNBr2. Whether this is due to the presence of only a single glutamate on MTX remains to be determined by comparing the location of its binding region with 5,10CHzH*PteGlu. Aside from establishing the amino acids associated with the binding regions for folate and its derivatives within a specific thymidylate synthetase, this study takes on an added dimension when comparing these binding parameters with enzymes from different sources. Thus as shown previously (20), conditions can be established where PteGlus markedly inhibits the Tzphage synthetase while barely affecting the Escherichia coli enzyme. It would therefore be of interest to determine whether the reason for this differential response is due to differences in the amino acid sequence associated with the folylpolyglutamate binding region of these enzymes or to some other factor. The techniques presented in this study should enable a solution to this problem to be derived. ACKNOWLEDGMENTS We would like to express our appreciation to Don U. Guarino and Judith Reid1 for their excellent technical assistance. REFERENCES 1. MCCUEN, B&him
R.
W. AND SIROTNAK, F. Biophya Acta 334.369-380.
M.
(1975)
AND
BAUGH
2. DoLNICK, B. J., AND CHENG, Y. C. (1977) J. Bid Chem 252.7697-7703. 3. KISLIUK, R. L, GAIJMONT, Y., BAUGH, C. M., GALIVAN, J. H., MALEY, G. F., AND MALEY, F. (1979) in Developments in Biochemistry (Kisliuk, R. L., and Brown, G. M., eds.), pp. 431-435, Elsevier/North-Holland, Amsterdam. 4. SCANLON, K. J., MOROSON, B. A., BERTINO, J. R., AND HYNES, J. B. (1979) Mel PhmmmL 16, 261-269. 5. SANTI, D. V., AND MCHENRY, C. S. (1972) Proc Nat. Ad sci. USA 69,1855-1857. 6. GALIVAN, J. H., MALEY, G. F., AND MALEY, F. (1976) Biochemistry 15,356-362. 7. GALIVAN, J. H., MALEY, F., AND BAUGH, C. M. (1976) Biochem Biophys Res Cmnmm 71, 527-534. 8. KISLIUK, R. L., GAUMONT, Y., LAFER, E., BAUGH, C. M., AND MONTGOMERY, J. A. (1981) Biu chf?tntitry 20,929-934. 9. PRIEST, D. G., AND MANGUM, M. (1981) Arch. Bie cherr Baaphys 210,118-123. lo. GALI~U, J., MALEY, F., AND BAUGH, C. M. (1977) Arch. Bicdmm Biophyg 134,346-354. 11. MALEY, G. F , BELLISARIO, R. L., GUARINO, D. U., AND MALEY, F. (1979) J. Bid C/z&m 254,13011304. 12. GALIVAN, J. H., MALEY, G. F., AND MALEY, F. (1975) Biochemistry 14.3334-3344. 13. KRUMDIEK, C. L., AND BAUGH, C. M. (1969) Bie chemistry 8,1568-1572. 14. WAHBA, A. J., AND FRIEDKIN, M. (1961) J. Bid C&-w. 236, PCll-12. 16. CHU, B. C. F., AND WHITELEY, J. (1977) Md Phar?rlad 13.80-88. 16. MALEY, G. F., BELLISARIO, R. L., GUARINO, D. U., AND MALEY, F. (1979) J. Bid Chem 254,12881295. 17. NIALL, H. D. (1973) in Methods in Enzymology (O’Malley, B., and Hardman, J. G., eds.), Vol 36, pp. 942-1010, Academic Press, New York. 18. LORENSON, M. Y., MALEY, G. F., AND MALEY, F. (1967) .X Bid Chem 242,3332-3344. 19. BELLISARIO, R. L., MALEY, G. F., GALIVAN, J. H., AND MALEY, F. (1976) Proc Nat Accd. Sci USA 73.1848-1852. 20. BELLISARIO, R. L., MALEY, G. F., GUARINO, D. U., AND MALEY, F. (1979) J. Bid Chem 254,12961300. 21. MALEY, G. F., MALEY, F., AND BAUGH, C. M. (1979) J. Bid Chem 254.7485-7487.
Studies on Identifying Lactobacillus GLADYS
F. MALEY,*
the Folylpolyglutamate Binding Sites of casei Thymidylate Synthetase’
FRANK
MALEY,*T~
AND
CHARLES
M. BAUGHT
*Division of Laboratories ad Research, New York State Department of Health, Albang, New York 12201; and the tDepartment of Biochemistry, College of Medicine, University of South Alabama, Mobile, Alabama 36688 Received
January
22, 1982
Pteroylheptaglutamate labeled with 14C in its proximal glutamate was activated with a water-soluble carbodiimide and reacted with Lactobacillus casei thymidylate synthetase in the presence and absence of dUMP. The latter nucleotide appeared to enhance the specificity of the reaction as the quantity of folylpolyglutamate fixed plateaued at 1.5 mol/mol of enzyme, but continued to increase above 2.0 in the absence of dUMP. The location of the covalently bound pteroylheptaglutamate was determined by first cleaving the labeled protein with cyanogen bromide and establishing that 80% of the radioactivity was present in the second of the five cyanogen bromide peptides of thymidylate synthetase. This peptide was then digested with chymotrypsin and the resulting peptides were purified by high-performance liquid chromatography. Two labeled peptides were isolated, with one being separated further into two pure peptides on gel exclusion chromatography. The three peptides represented residues 48-54, 52-61, and 55-61 of the synthetase, with the pteroylheptaglutamate fixed covalently to lysines 50 or 51 and 58. From the results presented, it would appear that each of the enzyme subunits is labeled, with the folylpolyglutamate being fixed to lysine 58 of one subunit and 50, 51 of the other.
Many, if not most of the antifolate drugs were designed originally to inhibit dihydrofolate reductase. However, it has been found that these compounds can also impair thymidylate synthetase (l-4), particularly in the presence of compounds which potentiate the binding of the folates such as the enzyme’s nucleotide substrate dUMP3 and its analogs (5, 6). Thus
the binding of PteGlu, H2PteGlu, 5,10CH2H4PteGlu, and folate analogs such as MTX is not detectable in the absence of dUMP or FdUMP but are bound in the presence of either nucleotide (5). Even in the case of the predominant forms of folate found in nature, the folylpolyglutamates, which bind to the synthetase in the absence of dUMP and related nucleotides, binding is greatly enhanced in the presence of the latter compounds (7). In view of these results it was not surprising to
i This investigation was supported in part by Public Health Research Grants GM26387 and GM26645 from the National Institutes of General Medical Sciences, PHS/DHHS. ‘Author to whom correspondence should be addressed. a Abbreviations used: dUMP, 2’-deoxyuridylate; FdUMP, 5-fluoro-2’-deoxyuridylate; PteGlu, pteroylglutamate (folate); HaPteGlu, dihydrofolate; loCH&PteGlu, lo-methyltetrahydrofolate; lo-CHOH4PteGlu, 10 - formyltetrahydrofolate; 5,10-CHa -
H,PteGlu,5,10-methylenetetrahydrofolate; PteGlua, pteroyltriglutamate; HaPteGlu,, dihydropteroylpentaglutamate; PteGlu7, pteroylheptaglutamate; MTX, methotrexate; CNBr, cyanogen bromide; TLCK, tosyllysine chloromethyl ketone; HPLC, high-performance liquid chromatography; TCA, trichloroacetic acid; PTH, phenylthiohydantoin. 551
0003-9861/82/080551-08$62.00/O Copyright All righta
8 1982 by Academic Press, Inc. of reproduction in any form reserved.
552
MALEY.
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find that the polyglutamate derivatives of 5,10-CHzH4PteGlu are considerably better substrates than 5,10-CHzH4PteGlu (8), a finding supported by the recent observation that the tightness of ternary deadend complexes of FdUMP-synthetase and the polyglutamates of 5,10-CHzH4PteGlu varies directly with the number of glutamates, up to five residues (9). Similarly, in those cases where protection of the enzyme against proteolytic degradation was measured the resistance to proteolysis was found to increase with the number of glutamates on folate (lo), an effect which implicates electrostatic interactions between the carboxyls of glutamate with a positively charged region of the enzyme. To complement our studies on identifying the nucleotide binding sites within the primary structure of thymidylate synthetase, which have revealed where FdUMP and most probably dUMP bind (ll), we have undertaken in this study to locate the region responsible for enhancing folylpolyglutamate binding. EXPERIMENTAL
PROCEDURE
Materials L cusei thymidylate synthetase was obtained from the New England Enzyme Center, Boston, Massachusetts, as a dialyzed ammonium sulfate fraction and purified to homogeneity as described previously (6). Folylpolyglutamates, including Pte[U-14C]GluGlus, which had a specific activity of 1 &i/pmol (1600 cpm/ nmol) were synthesized as described previously (12). Sephadex G-100 (loo200 mesh) and Sephadex G-25 (medium) were purchased from Pharmacia and BioGel filtration media were obtained from Bio-Rad. 1-Ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride was purchased from Pierce Chemical Company. TLCK-a-chymotrypsin was purchased from Worthington and TPCK-treated trypsin was kindly provided by Dr. T. H. Plummer, Jr. All other chemicals were of reagent grade or HPLC grade. Methods Fixation of Pte[U-‘4CJGluGlu+ Crystalline thymidylate synthetase was dissolved
AND BAUGH
in a minimum volume of 50 mM potassium phosphate, pH 7.1, 20 mM 2-mercaptoethanol and dialyzed against two changes of 2 liters each of the same buffer. The fully activated enzyme had a specific activity of 3.6 pmol/mg when assayed in the absence of Mgz+ and 5.3 pmol/mg when Mgz+ was present. The assay employed was a slight modification of the spectrophotometric procedure of Wahba and Friedkin (13). In each case the assay buffer was at pH 7.0. The activated enzyme was then passed over a column of Sephadex G-25, equilibrated with 50 mM potassium phosphate, pH 7.1, to remove the 2-mercaptoethanol from the enzyme solution. The resulting enzyme solution, 30 mg (0.43 pmol) in 3.0 ml, was mixed with 0.1 ml of 0.1 M dUMP. In a separate tube, 0.5 ml of 6 mM Pte[Ur4C]GluGluG (1600 cpm/nmol) was added to 0.5 ml of freshly prepared 10 mM l-ethyl - 3 - (dimethylaminopropyl)carbodiimide hydrochloride in 50 mM NaHC03, pH ‘7, similar to that described for the activation of methotrexate by Chu and Whiteley (14). The enzyme-substrate solution was then mixed with the labeled polyglutamatecarbodiimide adduct and allowed to react at room temperature in the dark. Small aliquots of 20 ~1 were removed at 30 and 60 min and assayed for binding with a nitrocellulose filter (5). The entire reaction was terminated at 60 min by the addition of 0.5 ml cold 50% TCA. After cooling, the resulting precipitate was centrifuged and the residue washed several times with 6 ml cold 5% TCA. An average yield of the polyglutamate bound adduct was 800 nmol of thymidylate synthetase monomer with 600 nmol of bound Pte[U-14C]GluGlus. All measurements of radioactivity were determined with a Packard Model 3320 Tri-Carb liquid scintillation counter using Aquasol as the counting medium. Chemical and enzptatic cleavage pm cedures. Thymidylate synthetase (29 mg) labeled with Pte[U-14C]GluGlus was cleaved with cyanogen bromide and the peptides were separated by extraction and column chromatography procedures as described previously (15). Labeled CNBr2 was subjected to chymotryptic hydrolysis at pH
FOLYLPOLYGLUTAMATE
AND L Casei THYMIDYLATE
8.2 to generate labeled peptides for compositional analysis. Reversed-phase high-perfiwmance liquid chromatography. Cyanogen bromide and chymotryptic peptides were purified by reversed-phase high-performance liquid chromatography with a Beckman Altex instrument containing a Waters PBondapak Cl8 column (0.4 X 25 cm). A linear gradient of 0.1% phosphoric acid to 90% acetonitrile/O.Ol% phosphoric acid was employed to effect the desired separations. In some cases, peptides were purified further by rechromatography on HPLC, followed by Bio-Gel-P2 column chromatography (1.5 X 85 cm) with 20 mM ammonium bicarbonate as the eluant. Amino acid analysis. Labeled protein and peptides were hydrolyzed for 24 h at 110°C in evacuated, sealed tubes containing 1.0 ml of constant boiling HCl. Single column amino acid analysis was performed on a Beckman Model 119CL amino acid analyzer. Edman degradaticms. Sequence determinations were performed with a Beckman Model 890 B automatic sequencer. Peptides were sequenced using N-alkyl-Ndimethylamine as the buffer (16). The released thiazolinone derivatives were identified after conversion to their respective PTH-amino acids by thin-layer chromatography and HPLC (15, 16). RESULTS AND DISCUSSION
Nature
of the Folate Binding
Site
When the effect of various folate analogs on the kinetics of 5,10-CHzH4PteGlu utilization is determined, it would appear that more than one site is involved in the binding of these compounds. Thus, as first shown with the chick embryo synthetase HzPteGlu is a noncompetitive inhibitor (17), which was shown subsequently to be the case for the human leukemic enzyme (2). In the latter instance MTX, PteGlus, and HzPteGlus were found also to be noncompetitive inhibitors of 5,10-CHzH4PteGlu while PteGlu, lo-CHsHIPteGlu, and lo-CHO-HIPteGlu appeared to be competitive inhibitors. On measuring the kinetics of interaction of MTX with the L casei synthetase, it too behaved as a non-
SYNTHETASE
mM-’
553
FHI
FIG. 1. Nature of the inhibition of thymidylate synthetase promoted by PteGlu7 and methotrexate. The reaction mixtures at 30°C contained 5 mM ascorbate, 50 mg 2-mercaptoethanol, (dl)t-H,PteGlu (FH,) as the variable substrate, 3.2 mM formaldehyde, 0.5 mM dUMP, 50 mM MgC12, and 50 mM Tris-HCl at pH 7.0. PteGlu7 and MTX were present at the indicated levels and the reactions were initiated by addition of enzyme. The spectrophotometric procedure of Wahba and Friedkin (13) was used to determine the initial velocity of each reaction.
competitive inhibitor as shown in Fig. 1. In contrast, it is clearly seen that PteGlu7 acts in a competitive manner, which appears to contrast with results obtained for the polyglutamates and the human synthetase (2). However, it is possible that the latter data are a consequence of only single time points being used to determine initial velocities, or possibly to an inherent difference in binding properties of the two enzymes. In any event it appears from the results in Fig. 1 that PteGlu, competes with 5,10-CHzH4PteGlu for its site on the L. casei synthetase, signifying that the pteroyl moieties overlap in their binding. Additional evidence favoring this thesis has come from nitrocellulose binding (5) in which the enzyme’s folate site was protected by first fixing 5,10-CHzH4PteGlu to the enzyme as part of the FdUMP ternary complex and then measuring the ability of [14C]PteGlu7 to bind. It was found that only about 20% of the PteGlu7 fixed to the complexed enzyme relative to the free enzyme. That which was bound to the ternary complex probably represents nonspecific binding. The greatly enhanced binding of the pteroyl polyglutamates relative to the monoglutamate is probably due to the greater opportunity for electrostatic interaction between the carboxyl anions of the polyglutamate residues and the lysyl
554
MALEY,
MALEY,
cations of a specific region of the protein. To identify this region, the carboxyls of the ultimate glutamate of PteGlu7 were activated, possibly to an (Y, y-anhydride, through the use of a water-soluble carbodiimide. The resulting anhydride could then covalently link to the c-amino groups of lysines in its proximity to facilitate the location of these residues within the primary sequence of the protein. Since dUMP enhances the binding of folate and its derivatives (6), including the folylpolyglutamates (7), probably by increasing their binding specificity, this nucleotide was included in the reaction solutions. If dUMP was not incuded, the binding of compounds such as PteGlu7 increased progressively with concentration to above 2 mol/mol of enzyme. However, in the presence of dUMP its binding plateaued at about 1.4 mol/mol of enzyme, a result which implies that the binding of PteGlu, is less random in the nucleotide’s presence (Fig. 2).
Identifying the PteGlu, Binding Site Once fixed to the enzyme, the location of PteGlu, was established by employing a procedure similar to that described for the FdUMP site (18). The labeled protein was cleaved with cyanogen bromide to its five peptides, which were purified by a combination of solvent extraction and BioGel column chromatography. From the
PteGlu,
ADDED
FIG. 2. Fixation of Pte[U-‘“C]GluGlus to thymidylate synthetase in the presence and absence of dUMP. The folylpolyglutamate was activated by a water-soluble carbodiimide and the degree of irreversible fixation was measured by a nitrocellulose filter assay (see Experimental Procedures for details).
AND
BAUGH TABLE
I
DISTRIBUTION OF RADIOACTIVITY IN THE CNBr PEPTIDES’ FROM L casei Pte[14C]GluGlus THYMIDYL.ATE SYNTHETASE CNBr peptide 1 2 3 4 5
Radioactivity (cpm/nmol)
Relative amount (%)
2 1225 49 225 4
0.1 81.4 3.2 15.0 0.3
a The CNBr peptides were isolated as described in (15). The peptides were further purified by reversedphase HPLC as described under Experimental Procedures.
distribution of 14C in the various CNBr peptides (Table I), it is obvious that most of the label (about 80%) is in CNBr2 as opposed to only 15% in CNBr4, the peptide to which FdUMP binds (15, 18, 19). That the peptide containing the folate was indeed CNBr2 was confirmed by amino acid analysis (Table II), which reveals ‘7 mol TABLE
II
AMINO ACID COMPOSITIONSOF CNBr2 AND PtdU-‘“C]GluGlus-CNBr2 Amino acid
Labeled CNBr2’
CNBrZb
Asp Thr Ser Glu” Pro GUY Ala Val Ile Leu 5r Phe LYS His Aw
a.7 3.9 3.8
9 4 4
15.9
9
3.6 5.3 2.6 2.6 2.7 9.0 1.4 7.8 7.2 5.5 3.8
4 5 3 3 3 9 2 8 7 6 4
a The values were obtained from 24-h acid hydrolysates and represent molar ratios relative to leucine. The specific radioactivity of purified labeled CNBr2 was 1266 cpm/nmol. b Composition of CNBr2 as determined previously (19). c Italics are used to emphasize increase in Glu residues from 9 to 15.9.
FOLYLPOLYGLUTAMATE
AND
L Casei THYMIDYLATE
TABLE
AMINO ACID COMWSITION Amino acid Thr Ser Glu Pro GUY Val Ile Leu Phe LYS
OF LABELED
Cl’ (nmol) 2.93 (1.51)
2
15.21 (7.34) 2.30 (1.19)
7
2.22 (1.14)
1
2.13 (1.10) 3.99 (2.01)
Position in CNBr2 cpm/nmol”
III
CHYMOTRYPTIC PEPTIDES FROM Pte[*%]GluGly Theor
1
1 2
C2 (nmol)
Theory
3.15 (1.26) 16.49 (6.60) 2.27 (0.91) 3.09 (1.24)
2.51 (1.00) 1.89 (0.76) 3.69 (1.48) 2.80 (1.12) 3.08 (1.24)
48-54 1110
555
SYNTHETASE
LABELED CNBr2
C3 (nmol)
Theory
1.59 (0.97) 11.95 (7.29)
1 8
1.77 (1.04)
I
1.43 (0.90)
1
2.87 (1.75)
2
2.13 (1.30)
I
52-61 1170
55-61
1130
’ Cl, C2, and C3 represent the three “C-labeled chymotryptic peptides in their sequential location in CNBr2. *The figures in parentheses are the estimated number of residues of each amino acid per peptide. For each peptide the number of nanomoles for all amino acids except Glu were added, and divided by the theoretical number of all amino acids except Glu to give an estimate of the number of nanomoles of the peptides (1.94 nmol for Cl, 2.50 nmol for C2, and 1.64 nmol for C3). The number of nanomoles of each amino acid was then divided by this value to give the estimated number of amino acid residues per peptide. ’ Specific radioactivities of the isolated peptides.
more of glutamate than expected for pure CNBr2, but in accord with that expected for a peptide containing PteGlu,. To further define the region occupied by PteGlq within CNBr2 this peptide was digested with chymotrypsin and the resulting peptide mixture separated by HPLC. From the elution profile presented in Fig. 3, it was apparent that most of the label was in two closely associated peaks. Further purification of these peptides on Bio-Gel-P2 resulted in the resolution of the slower eluting peptide into two peptides (C2 and C3). Amino acid analysis of the three purified peptides indicated that each contained 7 to 8 mol of glutamate/ mol of peptide (Table III). The fact that the number of glutamate residues in the isolated peptides differ by about one from theory is probably due to the use of an average value in calculating the number
A200
MINUTES
FIG. 3. Separation of chymotryptic peptides of CNBr2 labeled with Pt@-“CJGluGl~ by HPLC. The shaded areas represent regions where radioactivity was detected. The elution of the peptides was measured at both 236 and 210 nm. For additional details see Experimental Procedures.
MALEY,
556
MALEY.
of residues (Table III). Although peptides C2 and C3 each contained one lysine to which one equivalent of PteGlu7 was covalently linked Cl contained two tandem lysines, with only one apparently linked to PteGlu,, based on the molar ratio of glutamate to lysine. Sequence and amino acid analysis of each peptide revealed them to possess the indicated order of amino acids shown in Table IV, which reveals that chymotrypsin affected not only the anticipated cleavage between residues 54 and 55 (Phe-Gly) but also one that was unexpected, that between residues 51 and 52 (Lys-Val). Of interest are the specific radioactivities of the three peptides (Table III), which in each case reflects a 25% reduction in the specific activity of the starting material, PtflJ4C]GluGlus. This dilution was anticipated from the results in Fig. 2, which revealed that only about ‘75% of the enzyme had been labeled, a result which was confirmed by the finding that CNBr2 possessed the same specific radioactivity as the isolated chymotryptic peptides (cf. Tables I and III). Since the enzyme is composed of two identical subunits and the chymotryptic peptides were derived from both, it was not possible to distinguish between the labeling of one subunit by 1.5 mol of PteGlu, or the asymmetric labeling of each enzyme subunit by 0.75 mol of PteGlu,. Thus a situation can be envisaged TABLE PROBABLE
PEPTIDE
IV
SEQUENCES BY [“CjPteGlu,”
IN CNBr2
LABELED
(1) Thr-Thr-Lys-Lys-Val-Pro-Phe (2) Val-Pro-Phe-gy-Leu-Ile-Lys-Ser-C%Leu (3)
60 Gly-Leu-Ile-Lys-Ser-Glu-Leu
‘Peptide (1) represents the faster moving of the two labeled peaks in Fig. 3, while peptides (2) and (3) are from theslower moving peak, which was fractionated into these peptides on a Bio-Gel P2 column. Amino acid analysis (Cl, C2, and C3 in Table III) and partial sequencing established their location within the primary sequence of the synthetase as described under Experimental Procedures.
AND BAUGH TABLE
V
AMINO ACID COMPOSITION OF LIMITED TRYPSIN PEPTIDE CONTAINING Pte[U-“C]GluGlus Labeled peptide Amino acid
nmol
Residues”
Theoryb
Asp Thr Ser Glu Pro GUY Ala Val Ile Leu 5r Phe Tw LYS His Aw
8.66 7.95 5.39 21.66 6.99 8.61 0.35 3.10 5.11 19.3 0.00 10.57 ND 10.47 3.04 2.47
3.4 3.2 2.1 8.7 2.7 3.3 1.2 2.0 7.7 4.2 4.2 1.2 1.0
3 3 2 8” 2 3 1 2 7 4 1 4 1 1
o The values were obtained from 24-h acid hydrolysates and represent molar ratios relative to arginine. bThe composition of this limited trypsin peptide (residues 37-72) is based on a similar peptide isolated previously from CNBr2 (19). ‘Based on the one glutamate associated with this peptide plus the seven from PteGlu,.
where PteGlu, is fixed to residues (50, 51)4 and 58 of the same subunit resulting in a peptide specific radioactivity which is about 1.5 that of the initial specific radioactivity of PteGlu,, or one in which each subunit is labeled, but assymetrically (residues 50, 51 of one subunit and residue 58 of the other). In the latter case the specific radioactivity of the enzyme subunit, as reflected in CNBr2, would be 0.75 times the specific radioactivity of PteGlu7. If residues 48-61 could have been isolated as an intact peptide its specific radioactivity would have provided a solution to this problem, but since chymotrypsin had affected cleavages between residues 51 and 52 as well as between 54 and 55 it was not possible to obtain this peptide intact; nor ’ Since it is not possible at this time to distinguish which of the two lysines was labeled, both are listed.
FOLYLPOLYGLUTAMATE
AND
L Casei THYMIDYLATE
SYNTHETASE
557
SER-LYS-GLY-PHE-PRO-LEU cl
~EIJ LEU-TRP-PHE-LEU-H~~-GLY-ASP-~HR-A~PN-ILEILE-TR~-A~~-GL~-TR~-ALA-PHE-GL"-L~~-~~~-V~L-L~~-SER-A~~-GL"-TYR-~,~-G~Y-PRO-
A !f-
MET-THR-A~~-PHE-GL~-HIS-ARG-SER-GL~-~~~-A~~-P~O-G~"-P"E-A~~-AL*-V~L-TYR-~,~-
t f?-
GLU-MET-ALA-LVS-PHE-ASP-ASP-ARG-VAL-
5%I~s-Asp-ASP-ALA-PHE-ALA-ALA-LYS-TYR-
t f-
ASP-LEU-GLV-LEU-VAL-TYR-GLY-SER-GLN-
+%-ARG-ALA-TRP-His-THR-SWLYS-GLY-Asp-
+ !i-
ILE-ASP-GLN-LEU-GLY-ASP-VAL-ILE-GLU-
t So-IKE-LYS-THR-His-PRO-TYR-SER-ARG-ARG-~-
FIG. 4. The primary sequence of L casti thymidylate synthetase designating the active site region for FdUMP (area encompassing cysteine-198) and the region labeled by PteGlu, (area encompassing lysine 50, 51 and lysine 58).
was it possible to establish whether residue 50 was labeled in preference to 51 as a result of the inability of trypsin to cleave the Lys-Lys bond. In an effort to resolve the problem of which subunit was labeled by PteGluT, this compound was fixed to the intact enzyme as described in the methods, and the complex was subjected to citraconylation and trypsinization. The limited tryptic peptides were partially separated on a BioGel-P10 column and from those fractions containing 14C, a peptide was purified to homogeneity by HPLC. Amino acid analysis of this limited tryptic peptide (Table V) revealed that it most probably corresponded to residues 37-72 of the known sequence of the L. cusei synthetase (11). The specific radioactivity bf this peptide was 1400 cpm/nmol, which was as expected somewhat less than the specific radioactivity of the initial PteGlu,, indicating that only one PteGlu, had been fixed to each subunit. This peptide, which is
basically a mixture of residues 37-72 from each subunit, was then treated with chymotrypsin and the resulting peptides were isolated by HPLC as described in the methods and Fig. 3. Amino acid analysis and radioactivity determinations revealed that two of the chymotryptic peptides, encompassing residues 48-54 and 55-61, possessed the same specific radioactivity as the starting limited tryptic peptide, suggesting that the initial fixation of PteGlu, had been asymmetric. CONCLUSIONS
From the known sequence of CNBr2 (15), it is clear that the three chymotryptic peptides are derived from residues 48-61 of thymidylate synthetase as a result of cleavages at Lys-51, Phe-54, and Leu-61. The location of this region relative to that of the previously described FdUMP containing chymotryptic peptide is presented in Fig. 4.
558
MALEY,
MALEY,
Since the region to which the folate substrate or analog is fixed is obviously dependent on the length of the glutamate chain, the binding regions of other pteroylmono- and polyglutamates and their derivatives will possibly provide an important parameter for establishing their locus of action within the topography of the synthetase. Preliminary studies have shown that MTX, which is a noncompetitive inhibitor of 5,10-CHzH4PteGlu, in contrast to PteGlu7, binds to a different region of CNBr2. Whether this is due to the presence of only a single glutamate on MTX remains to be determined by comparing the location of its binding region with 5,10CHzH*PteGlu. Aside from establishing the amino acids associated with the binding regions for folate and its derivatives within a specific thymidylate synthetase, this study takes on an added dimension when comparing these binding parameters with enzymes from different sources. Thus as shown previously (20), conditions can be established where PteGlus markedly inhibits the Tzphage synthetase while barely affecting the Escherichia coli enzyme. It would therefore be of interest to determine whether the reason for this differential response is due to differences in the amino acid sequence associated with the folylpolyglutamate binding region of these enzymes or to some other factor. The techniques presented in this study should enable a solution to this problem to be derived. ACKNOWLEDGMENTS We would like to express our appreciation to Don U. Guarino and Judith Reid1 for their excellent technical assistance. REFERENCES 1. MCCUEN, B&him
R.
W. AND SIROTNAK, F. Biophya Acta 334.369-380.
M.
(1975)
AND
BAUGH
2. DoLNICK, B. J., AND CHENG, Y. C. (1977) J. Bid Chem 252.7697-7703. 3. KISLIUK, R. L, GAIJMONT, Y., BAUGH, C. M., GALIVAN, J. H., MALEY, G. F., AND MALEY, F. (1979) in Developments in Biochemistry (Kisliuk, R. L., and Brown, G. M., eds.), pp. 431-435, Elsevier/North-Holland, Amsterdam. 4. SCANLON, K. J., MOROSON, B. A., BERTINO, J. R., AND HYNES, J. B. (1979) Mel PhmmmL 16, 261-269. 5. SANTI, D. V., AND MCHENRY, C. S. (1972) Proc Nat. Ad sci. USA 69,1855-1857. 6. GALIVAN, J. H., MALEY, G. F., AND MALEY, F. (1976) Biochemistry 15,356-362. 7. GALIVAN, J. H., MALEY, F., AND BAUGH, C. M. (1976) Biochem Biophys Res Cmnmm 71, 527-534. 8. KISLIUK, R. L., GAUMONT, Y., LAFER, E., BAUGH, C. M., AND MONTGOMERY, J. A. (1981) Biu chf?tntitry 20,929-934. 9. PRIEST, D. G., AND MANGUM, M. (1981) Arch. Bie cherr Baaphys 210,118-123. lo. GALI~U, J., MALEY, F., AND BAUGH, C. M. (1977) Arch. Bicdmm Biophyg 134,346-354. 11. MALEY, G. F , BELLISARIO, R. L., GUARINO, D. U., AND MALEY, F. (1979) J. Bid C/z&m 254,13011304. 12. GALIVAN, J. H., MALEY, G. F., AND MALEY, F. (1975) Biochemistry 14.3334-3344. 13. KRUMDIEK, C. L., AND BAUGH, C. M. (1969) Bie chemistry 8,1568-1572. 14. WAHBA, A. J., AND FRIEDKIN, M. (1961) J. Bid C&-w. 236, PCll-12. 16. CHU, B. C. F., AND WHITELEY, J. (1977) Md Phar?rlad 13.80-88. 16. MALEY, G. F., BELLISARIO, R. L., GUARINO, D. U., AND MALEY, F. (1979) J. Bid Chem 254,12881295. 17. NIALL, H. D. (1973) in Methods in Enzymology (O’Malley, B., and Hardman, J. G., eds.), Vol 36, pp. 942-1010, Academic Press, New York. 18. LORENSON, M. Y., MALEY, G. F., AND MALEY, F. (1967) .X Bid Chem 242,3332-3344. 19. BELLISARIO, R. L., MALEY, G. F., GALIVAN, J. H., AND MALEY, F. (1976) Proc Nat Accd. Sci USA 73.1848-1852. 20. BELLISARIO, R. L., MALEY, G. F., GUARINO, D. U., AND MALEY, F. (1979) J. Bid Chem 254,12961300. 21. MALEY, G. F., MALEY, F., AND BAUGH, C. M. (1979) J. Bid Chem 254.7485-7487.