Study of thymidylate synthetase-function by laser Raman spectroscopy

19

Biochimica et Biophysica Acta, 391 (1975) 19--27 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 6 7 4 9 3

STUDY O F T H Y M I D Y L A T E SYNTHETASE-FUNCTION BY L A S E R RAMAN SPECTROSCOPY

a R A J E N D R A K. S H A R M A , a R O Y L. K I S L I U K , b S U R E N D R A P. V E R M A and b D O N A L D F.H. W A L L A C H

aDepartment of Biochemistry and Pharmacology, Tufts University School of Medicine, and bDivision of Radiobiology, Tufts-New England Medical Center, Boston, Mass. 02111 (U.S.A.) (Received N o v e m b e r 19th, 1974)

Summary The Laser-Raman spectra of thymidylate synthetase have been obtained with 488 nm excitation from an argon ion laser. Raman bands observed in the range 600--800 cm -~ have been assigned to functional groups of constituent amino acids. The band positions and intensities in the Amide I {1600--1700 cm -1 ) and Amide III (1200--1300 cm -1 ) regions, suggest that the enzyme is a mixture of s-helical and unordered conformations. L o w levels of ~-structure cannot be excluded. The spectra of the ternary complex formed by reacting thymidylate synthetase with (+)-L-methylenetetrahydrofolate and fluorodeoxyuridylate reveals a new band at 1618 cm -1 assigned to the C=N stretching vibration. This band may be due to formation of dihydrofolate or an iminium ion. The overall secondary structure of thymidylate synthetase does not change on formation of the ternary complex. However, the spectrum of the complex indicates local changes in groups such as ionized carboxyl (1400 cm -~), t r y p t o p h a n {1003 cm -~) and CH3, CH2 deformation modes (1440--1470 cm -1 ).

Thymidylate synthetase catalyzes the reaction of 5,10-methylenetetrahydrofolate with deoxyuridylate to form thymidylate and dihydrofolate [1]. H4-folate serves as the reductant to convert the methylene group to a methyl group which displaced the H on the 5 carbon of dUMP. The pyrimidine nucleotide analog 5-fluoro-2'~leoxyuridylate, a p o t e n t inhibitor of thymidylate synthetase, forms a ternary complex with the enzyme in the presence of methylenetetrahydrofolate [ 2--8 ]. Abbreviations: (-+)-L-H4-folate = mixture of diastereoisomers of 5,6,7,8 tetrahydrofolate at carbon 6; (+)-CH2-H4-folate = natural d i a s t e r e o i s o m e r of Ns,Nl0-methylenetetrahydrofolate; H2.folate = 7,8 d i h y d r o f o l a t e ; F d U M P = 5-fluoro-2'-deoxyulidylate.

20 Spectrophotometric studies [2,6--8] indicate that H4-folate is chemically altered upon formation of the ternary complex. Comparison of the difference spectrum between H4-folate and H2-folate [2] with the spectral change occurring upon formation of the ternary complex suggests that the new folate species might be H2 -folate. Another possible interpretation is that an iminium ion (N*=CH2) is formed [9,10]. We report here the Raman spectra of thymidylate synthetase and ternary complex which yield information on the structure of the protein and the nature of the tetrahydrofolate present in the complex. Experimental Materials Folic acid (Nutritional Biochemicals) was used to prepare (+)-L-H4-folate by catalytic hydrogenation [11] over platinum catalyst. (+)-L-H4-folate was prepared enzymatically from dihydrofolate by the method of Mathews and Huennekens [12], as modified [13]. FdUMP was obtained from the TerraMarine Bioresearch. Hydroxylapatite was purchased from Bio Rad Laboratories. Methods Enzyme preparation Crude bacterial extracts containing thymidylate synthetase were prepared at the New England Enzyme Center [14] from a methotrexate-resistant strain of Lac to bacillus casei developed in this laboratory [ 15 ]. Thymidylate synthetase was purified according to the method of Leary and Kisliuk [16] through the hydroxylapatite chromatography step. The pooled fractions were concentrated [16] and dialyzed overnight against 80 mM potassium phosphate, 100 mM KC1, 1 mM disodium EDTA, pH 6.8. Further purification was obtained by gel filtration on Sephadex G-100. Approx. 80--100 mg protein in 4--6 ml was applied to the column (2.6 X 86) and eluted with the same solution. This method was developed by Dr Richard Leary in this laboratory. Fractions with specific activities of 126--145 were pooled and concentrated by ammonium sulfate precipitation, dialyzed as above and the gel filtration procedure was repeated. This protein (yield 26 mg) showed a single band when 180 pg were subjected to acrylamide gel electrophoresis at pH 8.5 [17]. The extinction coefficient of enzyme at 280 nm was taken as 108 000 based on amino acid analyses, performed by Dr B.H. Davis and Dr J. Ozols of the University of Connecticut. Tryptophan analysis of the enzyme by magnetic circular dichroism [18] was carried out by Dr Barton Holmquist (Biophysics Research Laboratory, Harvard Medical School, Boston, Mass.) showed 12 tryptophan residues per Ms 68 000. Enzyme assay Thymidylate synthetase activity was measured spectrophotometrically [19]. Specific activity is defined as the number of micromoles of thymidylate formed per hour per mg of protein at 30 ° C. Raman spectroscopy Aqueous solutions of thymidylate synthetase 1.8 • 10-4 M, (+)-CH2-H4-

21 folate 4.5 • 10 -4 M, FdUMP 4.5 • 10- 4 M and H2 -folate 4.5 • 10 -4 M alone or in various combinations, were transferred to 0.9--1 m m ID Kimex capillaries. After sealing, the sample capillaries were placed in a Miller-Harney cell [20] for t e m p e r a t u r e control. The t e m p e r a t u r e was regulated at 20 ° + 2 ° by a flow of nitrogen regulated by a t e l e t h e r m o m e t e r . T e m p e r a t u r e cont rol was checked by melting p o in t measurements on standard lipids in the laser beam. Raman spectra were recorded with a Ramalog 4 Raman s p e c t r o m e t e r (Spex Industries, Metuchen, N.J.) interfaced to an Interdata C o m p u t e r (Model 70). An argon-ion laser (Spectra Physics model 164), tuned at 488 nm, was used as an excitation source, generally at 100 mW power. The Raman scattering was d etected by a thermoelectrically cooled photomultiplier (RCA) and was recorded in terms of photons/s. The " d a r k " counts of the p h o t o cell were < 1 0 0 counts/s and " l i ght " counts were of the order of 104 counts/s. The scanning was d o ne through the c o m p u t e r at frequency intervals of 1 cm -~/s. The dwell time at each f r e que nc y step was maximally 1 s. The p h o t o n counts were stored in the c o m p u t e r m e m o r y during scanning (2--3 scans). The averaged, stored, spectra was then pl ot t e d as on the Ramalog recorder after adjustm e n t of plotting parameters (see figure legends). The time required to obtain spectra f r o m 500--1800 cm -~ was approx. 22 min. We kept the e n z y m e in the laser light for ab o ut 5--10 min before scanning. This time is sufficient for a constant baseline which otherwise varies due to fluorescence. Carbon tetrachloride was used to check f r e q u e n c y calibrations. The e n z y m e retained its original activity after Raman spectroscopy.

Infrared spectroscopy Aqueous solutions of the e n z y m e or the constituents of the ternary complex were deposited on AgC1 plates, frozen and lyophilized. Infrared spectra were recorded at 30°C and 55% relative humidity, using a Perkin-Elmer 621 spectrophotometer. Results and Discussion

The Raman and infrared spectra o f thymidylate synthetase A computer-averaged plot of the t h y m i d y l a t e synthetase Raman Spectrum is represented in Fig. 1. Spectral assignments based on r e p o r t e d Raman frequencies o f different proteins and polypeptides are given in Table I.

Amide I and Amide III regions The Amide I vibrations, due to the peptide linkages o f proteins and polypeptides ( p r e d o m i n a n t l y C=O stretching) yield bands between 1600 cm -1 and 1700 cm -1 . T h y m i d y l a t e synthetase exhibits three bands in this region. These center at 1630 cm -~ , 1655 cm -~ and 1680 cm -1 , but are som ew hat diffused by the broad water peak centered at 1640 cm -~ . The positions of Amide bands are k n o w n to depend on the secondary structures o f the peptide linkages [ 2 1 ] . Thus, a-helical poly-L-lysine in aqueous solution is characterized by a very strong Amide I band at 1647 cm -~ , whereas the anti-parallel fl-structure of this p o l y p e p t i d e exhibits a major Amide I peak at 1670 cm -~ [ 2 1 ] . With aqueous solutions of " u n o r d e r e d " poly-L-lysine one observes three Amide I bands at 1653 cm -~ , 1665 cm -~ , and 1683 cm -~ [ 2 2 ] .

22

~1 ~

/'x

~

~\~

o

°

/

J I 1800

I

I 1600

I

I 1400

I

I 1200

I

I I000

I

I ,BOO

|

I 600

Fig. 1. R a m a n s p e c t r u m of t h y m i d y l a t e s y n t h e t a s e ( 1 . 8 ' 10 -4 M) in 0 . 0 8 M p o t a s s i u m p h o s p h a t e , 0.1 M KC1 and 0 . 0 0 0 1 M E D T A ( d i s o d i u m salt) c o n t a i n i n g 0.1 M 2 m e r c a p t o e t h a n o l , p H 6.8. T h e l o w e r spectrum is of t h e s a m e s o l v e n t a l o n e . O r d i n a t e full scale 7.0 " 1 0 4 p h o t o n s / s . Abscissa represents frequency shift (A c m - 1 ) from laser excitation frequency 4 8 8 . 0 n m ( 2 0 4 9 2 c m - 1 ) ; p o w e r 1 0 0 roW; plotting step = 50 c m - 1 / s ; r e s o l u t i o n 8 c m - 1 ; t e m p e r a t u r e 2 0 ° C .

This conformational sensitivity of the Amide I region should, in principle, allow a conformational analysis of t h y m i d y l a t e synthetase but, at the protein concentrations feasible here the large scattering contribution of water relative to the Amide I scattering intensity makes assignment of band positions difficult and precludes extensive conclusions. However, the lack of a sharp 1670 cm -1 peak suggests that the fl-structure is not d o m i n a n t in thymidylate synthetase. This is consistent with the infrared spectra. Our spectra do not allow an evaluation of the relative proportions of helical and " u n o r d e r e d " structure. To eliminate water interference we lyophilized t h y m i d y l a t e synthetase and recorded the Raman spectra of the enzyme redissolved in 2 H2 O. (Fig. 2). The Amide I peaks (Amide I') now lie at 1630 cm -1 and 1665 cm -1 . No band is observed at 1658 cm -1 , the frequency characteristic of H-structured poly-Llysine in 2 H2 O solution [21]. The 1630 cm- 1 and 1665 cm- 1 bands of thymidylate synthetase are, however, consistent with a mixture of a-helical and "unordered" peptide, since the principal Amide I bands of a-helical and "unordered" poly-L-lysine in 2 H2 O lie at 1632 cm -1 and 1660 cm -1 [21]. Infrared spectra (Amide I region) of lyophilized t h y m i d y l a t e synthetase films (not shown) show a single, strong band maximal at 1650 cm -~ , lacking irregularities at 1630--1640 cm -~ , the absorption frequency of the ~-structure [23]. Since pure helical polypeptides absorb maximally at 1650--1655 cm -1 and the " u n o r d e r e d " conformation yields an Amide I peak at 1656 cm -~ , we cannot distinguish between these conformations. The data are consistent with the conclusions drawn from the Raman spectra, that t h y m i d y l a t e synthetase is primarily a mixture of helical and " u n o r d e r e d " peptide arrays. The Amide I region of t h y m i d y l a t e synthetase also exhibits bands not of peptide origin. Thus, the 1608 band (2 H2 O; Fig. 2) probably arises from Trp and the peaks at 1712 cm -~ and 1735 cm -1 derive from unionized carboxyl groups (C=O stretch).

23 TABLE I FREQUENCY ASSIGNMENTS OF THYMIDYLATE SYNTHETASE, TERNARY COMPLEX (THYMID Y L A T E S Y N T H E T A S E + ( + ) - C H 2 - H 4 - F O L A T E + f d F U M P ) A N D ITS C O N T R O L S N u m b e r s in p a r e n t h e s i s r e f e r t o t h y m i d y l a t e s y n t h e t a s e in 2 H 2 0 Frequency (cm -1) Thymidylate synthetase

(+)-CH 2 - H 4 - f o l a t e

Thymidylate synthetase + (+)-CH2-folate

Ternary complex

H2-folate

(1735) (1712)

ester

1680 (1665) 1655 1630

Tentative assignment

1660

1678 1660

1665

1618

1620

1655 1630

(1630) 1620

amide I and H20 v(C=N)

(1608) 1555

(1553) (1540)

1555

1558

Trp

1530 (1512) 1450 1400

(1455) (1403) (1385)

1480 1440

1470

1485

1430 1400

1430

1360

1358

1455 1410

CH 2 deformation COO-

1380

1348

1340 (1315)

T r p a n d C-H d e f o r mation

1310

1303 1293

1280 1260

(1280)

1210 1130 1082 1.003 99O

1280 Amide Ill 1245

1250

1200

1210 1120 1080

1090 1015

1240

1240 1185 Tyr and Phe

1070 1020

1075

P(C-N) Trp

950

u(C-C)

88O 850

Tyr and Trp

760 640

Trp v(C-S)

The Amide III vibrations {mainly C-N stretching plus N-H deformation) yield scattered peaks between 1200 cm-1 and 1300 cm -1 . T h y m i d y l a t e synthetase exhibits two weak rather broad bands in this region at 1260 cm- 1 and 1280 cm -1 . {Fig. 1). These again do n o t permit detailed conformational analyses [24--26].

Ring vibrations The strong bands at 1608 cm -l (2 H2 O) (Fig. 2) and 1003 cm -1 (Fig. 1) are attributable to the indole rings of the twelve t h y m i d y l a t e synthetase trypto-

24 o

V~ 0

Thym~dylole syn,hetose

I 1800

i

i 1600

I

i 1400

I

I 1200

Fig. 2. R a m a n s p e c t r u m o f t h y m i d y l a t e s y n t h e t a s e ( 1 . 8 • 1 0 .-4 M) i n 2 H 2 O o O r d i n a t e f u l l scale 6 . 0 . p h o t o n s / s . O t h e r c o n d i t i o n s w e r e t h e s a m e as i n F i g . 1.

10 4

phans (see M e t h o d s ) . These residues also a c c o u n t for the bands at 1 5 5 5 c m -~ , 1 3 4 8 c m -1 , 8 8 0 c m -~ , and 7 6 0 c m -~ (Table I). The 880 c m -~ b a n d c a n n o t be securely assigned since SH d e f o r m a t i o n (cysteine) p r o d u c e s a peak at 875 c m -~ . T h e p - h y d r o x y p h e n y l ring o f t y r o s i n e also yields a characteristic b a n d at 8 8 0 c m -! .

C-H deformation modes (1300--1500 cm -~ ) The R a m a n s p e c t r u m o f t h y m i d y l a t e s y n t h e t a s e b e t w e e n 1 3 0 0 c m -~ and 1 5 0 0 c m - ' resembles t h a t o f b o v i n e s e r u m a l b u m i n [ 2 5 ] . The strongest CH d e f o r m a t i o n b a n d lies at 1 3 4 8 c m - ' . T h e b r o a d b a n d b e t w e e n 1 4 4 0 cm - ' and 1 4 7 0 c m -~ is a t t r i b u t e d t o the CH2 and CH3 d e f o r m a t i o n m o d e s . T h e b r o a d s h o u l d e r near 1 4 0 0 c m -~ m a y be d u e t o ionized c a r b o x y l groups.

C-N and C-C stretching (900--11 O0 cm -' ) T h y m i d y l a t e s y n t h e t a s e exhibits a s t r o n g C-N s t r e t c h i n g b a n d at 1 0 8 2 c m - ' ; as discussed b e l o w this varies with e n z y m e " s t a t e " . The bands at 9 9 0 c m -~ and 9 5 0 c m - ' are assigned t o C-C s t r e t c h i n g vibrations.

C-S stretching and S H deformation (600--900 cm-I ) T h y m i d y l a t e s y n t h e t a s e c o n t a i n s f o u r residues o f cysteine a n d 12 residues o f m e t h i o n i n e w h i c h m a y c o n t r i b u t e t o R a m a n scattering in this region. We observe f o u r distinct peaks b e t w e e n 6 0 0 c m -1 a n d 9 0 0 c m - ' , at 880 c m ~1 , 8 5 0 c m ~1 , 7 6 0 c m - ' and 6 4 0 c m - ' . The peaks at 8 5 0 c m -1 and 760 c m - ' are a t t r i b u t a b l e to the ring vibrations o f Trp. As n o t e d , the p e a k at 8 8 0 c m - ' c a n n o t be u n a m b i g u o u s l y assigned since b o t h the S H - d e f o r m a t i o n v i b r a t i o n o f cysteine and a ring vibration o f a r o m a t i c residues p r o d u c e R a m a n scattering at

25 this frequency. Since there are 38 residues of Trp + Tyr and only 4 residues of Cys SH, we are inclined to assign the 880 cm -~ band to the aromatic residues. The C-S stretching bands of cysteine lie between 690 cm -1 and 680 cm -1, and those of Met at 724 cm- ~, 701 cm- ~ and 655 cm- l [26]. The shoulder in the thymidylate synthetase spectrum at approx. 690 cm-1 might thus be assigned to C-S stretching. We suspect that the 640 cm- 1 band is also due to Met since C-S stretching bands can vary widely in frequency depending on the micro-environment of the C-S linkage [ 27 ].

Differences in Raman scattering between thymidylate synthetase and the ternary complex FdUMP does not yield a resolved Raman Spectrum, at the concentrations used. Mixtures of thymidylate synthetase and (+)-CH2-H~-folate, or of thymidylate synthetase and FdUMP yield spectra identical to that of thymidylate synthetase without additions. However, the spectrum of the ternary complex (enzyme + CH2-H4-folate + FdUMP) is distinctly different from that of thymidylate synthetase in the following respects (Fig. 3):

•

o

T,~ CH2THF

m °

H

-~

~

T?

I

2 .2

\j

o

H2

N5,NI~CH~THF co ,o co

--

H

"2"

"2.2 .~, ,YI1 6.kICC~1o .

synthetaselse/ '

'J

\I v~

C H 2 T H F/

T,,.,

.--

~ -A i v

-- ~

~ L

o

~

~

j

I

O

o

e4

DHF H

.. I

1800

I

I

1600

I

I

1400

I

I

1200

I

H2 s~s,~6 l 22 C~ ~o

~ N-R

CH 2

I

1000 Irninium ion form of THF

F i g . 3, R a m a n s p e c t r u m o f t e r n a z y c o m p l e x (CH2THF), 4.5 ' 10-4 M + FdUMP 4.5 • 10 -4 ( 4 . 5 • 1 0 --4 M ) i n t h e s a m e b u f f e r as u s e d i n (+)-CH2-H4-folate ordinate full scale = 3 " 103

(thymidylate synthetase 1 . 8 • 1 0 - 4 M + ( + ) - C H 2 - H 4 - f o l a t e M), H24olate (DHF), (4.5 • 10-4 M) and (+)-CH2-H4-folate F i g . 1. O t h e r c o n d i t i o n s w e r e s a m e as in F i g . 1, e x c e p t i n photons/s.

F i g . 4. S t r u c t u r a l f o r m u l a o f C H 2 - H 4 4 o l a t e (NsN 10-CH2-THF), i m i n i u m i o n c o u l d a l s o o c c u r o n t h e N 10 p o s i t i o n .

H2-folate

(DHF)

and iminium

ion. The

26 1 . A new band appears at 1618 c m -~ . 2. The 1082 cm -1 band of t h y m i d y l a t e synthetase disappears and the intensity o f the 1003 c m - ' Trp peak decreases markedly. 3. The peak at 1450 cm -~ and shoulder at 1400 cm -~ are replaced by three bands at 1470 cm -1 , 1430 cm -~ and 1400 cm -~ . The position of the 1618 c m - ' band is characteristic of the C=N stretching vibration [ 2 8 ] . In support of this assignment is the fact t hat CH2 H4-folate, which has a C=N bond (Fig. 4) exhibits a weak Raman band at 1618 c m - 1 - - 1 6 2 0 cm -~ (Fig. 3). In H2-folate which has an additional -C=N- bond per molecule (Fig. 4), the 1618 cm -~ band is sharp and intense (Fig. 3). One could thus assign the 1618 cm -1 band of the ternary com pl ex to the form at i on of H2-folate. However, the 1618 cm -1 band in the complex appears rather broad c o mp ar ed with t hat of H2-folate. One should therefore consider possible f o r m a t i o n o f an iminium ion [ 9 , 1 0 ] . The C=N ÷ vibrations of iminium ions yield Raman scattering bands at a ppr oxi m a t el y the same frequency as C=N vibrations [ 2 9 ] , but p r o t o n a t i o n o f the N a t o m generally causes a reduct i on of scattering intensity as well as considerable band broadening [ 3 0 - - 3 2 ] . Our data are thus compatible with the suggestion t ha t an iminium ion derivative rather than H : - f o l a t e is the folate intermediate in the ternary complex. However, we cannot clearly discriminate between the two possibilities at this stage. The remaining alterations at 1003 cm -~ , 1082 cm -' and between 1400 crn-~ and 1500 cm-1 are most reasonably attributed to structural alterations within t h y m i d y l a t e synthetase. This is particularly true for the reduction in intensity o f the 1082 cm -~ and 1003 c m - ' bands and for the appearance of the 1400 cm -~ peak. The latter is most reasonably attributed to an increase in the p r o p o r t i o n o f ionized carboxyls. Whatever the structural modifications in the enzyme, however, we see no spectral alterations indicative of a change in secondary structure. Acknowledgement

S u p p o r ted by Grants # CA-13061 and GB-32133 from the U.S. Public Health Service and the National Science F o u n d a t i o n , respectively (S.P.V. & D.F.H.W.) and award PRA-74 from the American Cancer Society (D.F.H.W.) and by Grant CA10914 f r om the National Cancer Institute (R.L.K.).

References 1 2 3 4 5 6 7 8 9 10 11

F r i e d k i n , M.E. ( 1 9 7 3 ) Adv. E n z y m o l . 38, 2 3 5 - - 2 9 2 S h a r m a , R.K. a n d Kisliuk, R . L . ( 1 9 7 3 ) Fed. Proc. 32, 591 Santi, D.V. a n d McHenxy, C.S. ( 1 9 7 2 ) Proc. Natl. Acad. Sci. U.S. 69, 1 8 5 5 - - 1 8 5 7 L a n g e n b a c h , R.J., D a n e n b e r g , P.V. and Heidelberger, C. ( 1 9 7 2 ) B i o c h e m . Biophys. Res. C o m m u n . 48, 1565--1571 Aull, J . L . , L y o n , J . A . a n d D u n l a p , R.B. ( 1 9 7 4 ) M i c r o c h e m . J. 19, 2 1 0 - - 2 1 8 Santi, D.V., M c H e n r y , C.S. and S o m m e r , H. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 4 7 1 - - 4 8 1 D a n e n b e r g , P.V., L a n g e n b a c h , R , J . and Heidelberger, C. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 9 2 6 - - 9 3 3 S h a r m a , R.K. a n d Kisliuk, R.L. ( 1 9 7 4 ) Fed. Proc. 33, 1 5 4 6 Benkovic, S.J., B e n k o v i c , P.A. a n d C o m f o r t , D.R. ( 1 9 6 9 ) J. A m . C h e m . Soc. 9 1 , 5 2 7 0 - - 5 2 7 9 Kallen, R . G . and Jencks, W.P. ( 1 9 6 6 ) J. Biol. Chem. 2 4 1 , 5 8 5 1 - - 5 8 6 3 Kisliuk, R.L. ( 1 9 5 7 ) J. Biol. C h e m . 2 2 7 , 8 0 5 - - 8 1 4

27 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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