Vol. 83, No. 4, 1978

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

August 29,1978

Pages

"F

NMR OF THE 5-FLUORODEOXYURIDYLATE-THYMIDYLATE BINARY COMPLEX Charles

A. Lewis,

Jr.,

Paul

D. Ellis,

1509-1517

SYNTHETASE

and R. Bruce

Dunlap

Department of Chemistry University of South Carolina Columbia, South Carolina 29208 Received

July

19,

1978

SUMMARY: Formation of the 5-fluorodeoxyuridylate-thymidylate synthetase binary complex generates a 19F nmr resonance 1.3-1.4 ppm to hiqher shielding from free ligand, probably as the result of rotation'& the pyrimidine ring about the glycosyl bond. Addition of sodium dodecyl sulfate to the complex produces the spectrum of free ligand indicating that in contrast to the ternary complex of enzyme:nucleotide:cofactor, the binary complex does not contain a covalent bond linking the nucleotide In the presence of a 2.5 molar excess of nucleotide, 1.55 to the enzyme. moles were bound per mole of enzyme in Tris-Cl buffer. Under comparable conditions in sodium phosphate, 0.64 moles were bound, suggesting a specific buffer effect by phosphate. The synthesis reductively both

the

potently peutic

single

(I)

is

carbon

inhibited

of

unit

believed

(3).

activity of

synthetase,

CH2H4folate

as the

source

(1,2).

This

equivalents

is derived Numerous

FdUMP and CH2H4folate

complex

by thymidylate

and reducing

by FdUMP which

of enzymatic ternary

performed

dUMP, employing

5-fluorouracil

interactions

covalent

dTMP is

methylates

agent

inhibition

of

with

results

from studies the

from

the

have

enzyme

analogous

deoxyribose

to the

of enzyme

is

chemothera-

examined

to

formation

FdUMP:enzyme:CH2H4folate

to be exactly

cancer

which

determine

the that

of a stable (4-7).

catalytically

This

complex

competent

- PO,

I Abbreviations used: CH,H,folate, dTMP, thymidine 5'-monophosphate; FdUMP, 5-fluoro-2'-deoxyuridylate;

(+)-5,lOmethylenetetrahydrofolate; dUMP, 2'-deoxyuridine 5'monophosphate; SDS, sodium dodecyl sulfate 0006-291X/78/0834-1509$01.00/0

1509

Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

Vol. 83, No. 4, 1978

complex

BIOCHEMICAL

involving

dUMP.

C-5 hydrogen

produces

freezing

enzyme

the

variety

of

cysteine

sources

thiol

inhibitory,

to

However,

the

spectral

the

folate

at

a point

initiate

substitution

carbon

properties and enzyme

ternary

complex

events

leading

to

is

6 of

for

of complexes,

both

completed

formation binary

the

to the

(8,9).

pyrimidine

in this

and elucidated linkages

the

ring

laboratory

relative

to more

ternary

ring fully

complex,

FdUMP:thymidylate

for

Evidence

from

an enzyme

bound

catalytic

and

a

(2). have

determined of

in the

intact

describe

the

we have

synthetase

the thus

stereochemistry

pyrimidine

In order of

path.

requirement

the

fluorine

to cleavage,

reaction

the

RESEARCH COMMUNICATIONS

of

resistant

the

formation

" F nmr studies

of the

along

has demonstrated

native

nmr studies

the

a C-F bond which

by attacking

Recent

AND BIOPHYSICAL

initiated

19F

complex.

EXPERIMENTAL: Thymidylate synthetase was isolated in the presence of exogenous thiol from amethopterin resistant Lactobacillus casei according to the procedure of Lyon -et al. (10). Enzyme for studies ins-Cl was subjected to column chromatography on DEAE-Sephadex to remove tightly bound phosphate (9). Roughly 30 mg of purified enzyme was concentrated to a volume of 4.5 ml to yield a final concentration of about 80 uM. The enzyme was activated by dialysis in 2 liters of 0.1 M buffer (Tris-Cl or sodium phosphate) at pH 7.4 containing 1 mM EDTA and 25 mM 2-mercaptoethanol and was then dialyzed against two successive 200 ml volumes of the same buffer containing 33% D20 to provide a lock signal for the nmr spectrometer. Immediately prior to preparation of the nmr sample, the enzyme was assayed and found to possess a specific activity of 3.2-3.3 units/mg (11). Binary complex samples were prepared in 18 mm nmr sample tubes by mixing the enzyme solution with a 2.5 fold molar excess of FdUMP, which had been synthesized and chromatographed according to the procedure of Dawson --et al. (12). 19 F nmr spectra were obtained at 94.1 MHz on our highly modified Varian XL-loo-15 nmr spectrometer utilizing the 18 mm multinuclear probe (13). All data were obtained at 20 + 1 'C in the Fourier transform mode using 20 psec pulses (60" flips) witli 0.4 set acquisition time, 0.2 set pulse delay, acquiring 2K data points and transforming 8K, with 0.3 hz exponential broadening. All spectra were referenced to FdUMP dissolved in the buffer used in the experiment, which is observed about 88.8 ppm to higher shielding of trifluoroacetic acid in ~~0. After obtaining spectra of the native complexes, the appropriate amount of a 20% aqueous solution of SDS (electrophoretic grade) was added to the nmr sample to a final concentration of 1.2%, which had been shown to denature the ternary complex (9). In each case, spectra of the denatured complexes were compared to buffered solutions of FdUMP containing 1.2% SDS. RESULTS: and in the

Figures binary

1 and 2 illustrate complex

with

the

thymidylate

1510

"F

nmr spectra synthetase,

of native

FdUMP, free and

Vol. 83, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

t-(

1 PPrn

I

1500

Figure

denatured. higher

1

I

1000

Hz

c

500

0

1. The 94.1 MHz 19 F nmr spectra of FdUMP and the binary complex formed with thymidylate synthetase in 0.1 M Tris-Cl at pH 7.4. (A) 1.43 mM, FdUMP, 2000 pulses (B) 79 PM thymidylate synthetase in the presence of a 2.5 molar excess of FdUMP, 100,802 pulses (C) same as B with 1.2% SDS, 57,324 pulses (D) 1.43 mM FdUMP with 1.2% SDS, 2,000 pulses.

The binary shielding

of

complex FdUMP.

exhibits

A small

a resonance resonance

1511

appears

1.3

to 1.4

between

ppm to the

two

Vol. 83,No.4,

BIOCHEMICAL

1978

2500

2000

AND BIOPHYSICAL

lsoo

RESEARCH COMMUNICATIONS

500

1000

0

Hz

Figure

2. The 94.1 MHz " F nmr spectra of FdUMP and the binary complex formed with thymidylate synthetase in 0.1 M NaP04 at pH 7.4. (A) 1.43 mM FdUMP, 5.528 pulses (B) 84 PM thymidylate synthetase in the presence of a 2.5 molar excess of FdUMP, 118,333 pulses (C) 81 $l enzyme in the presence of a 5.0 fold molar excess of FdUMP, 105,668 pulses (D) same as C with 1.2% SDS, 66,327 pulses.

larger

peaks

material.

and appears The spectral

to be due to parameters

of

some non-specifically the

1512

binary

complex

bound are

contrasted

Vol. 83, No. 4, 1978

Table

Composition

BIOCHEMICAL

I.

of

AND BIOPHYSICAL

19 F NMR Spectral Parameters of and Ternary Complexesa

Comparison Binary

Complex

Buffer

FdUMP:Thymidylate Synthetase

RESEARCH COMMUNICATIONS

0.1

State

M Tris-Cl pH 7.4

Chemical Shift ppmb

Thymidyl%t$ FdUMP:CH H folate: Synthetase

aTernary

complex

35c

+0.2

14c

Native

-1.43

15c

Denatured

+0.1

15c

Native

-12.4

96

Denatured

-1.9

65

bChemical negative

shifts shifts

'Linewidths enzyme.

of

with

buffer,

ternary

complex

in Tris-Cl

are

is

strikingly indicated

the

dependent, the

binary

same 2.5

Doubling amount

percentages

fold

constant of

the

of the

addition

extent

buffer,

ligands

in the

The broader

with

absence

linewidths

contaminating

of SDS appears

of binary

complex

forms

formation excess

of

observed

bound fluorine

I.

due to metals

corroborating

FdUMP excess

of material

free

appropriate

the

to remove

the

FdUMP.

the

The diminished

dissociation

the

that

to

in Table

FdUMP; the with

observed

comparable

apparently

interaction

that

buffer. at

the

buffer

(8).

are in ppm from FdUMP in the to higher shielding.

the

of

from It

Byrd

are

chelating

metal

from

complex

those

observed

M NaP04 pH 7.4

0.1 pHM NaP04 6.8

data

Linewidth hz

-1.25

Native Denatured

0.1

of

of

earlier to

binary

the

in this the

nucleus

from sample enzyme. present

1513

formation

studies greatest

complex

FdUMP may reflect going

to

complex

is

(14,15), extent

in Tris-Cl

in phosphate

the

Tris-Cl

to

did

lead

tripling

buffer of

phosphate

the (14).

to an increase

Calculation in each

which

species

of

the permitted

in

of

BIOCHEMICAL

Vol. 83, No. 4, 1978

Table Buffer

II.

Composition

FdUMP excess

AND BIOPHYSICAL

and Binding

% Free

Ratios

of

% Non-Specific

RESEARCH COMMUNICATIONS

Binary

Complexes

% Binary

Complex

moles FdUMP moles enzyme

Tris-Cl

2.5

25.0

12.8

62.2

1.55

Nap04

2.5

68.1

6.4

25.5

0.64

5.0

75.3

6.1

18.6

0.93

calculation

of

nonspecific

binding

can only

the

binding

discriminate

DISCUSSION:

The ability

or lower of

binding

19

by

shown

F spectrum

between

of

complex

19

in the

overestimations

binary

ratios

bound

F nmr permits

(161.

It

results

from

cysteine charge

might the

the

have

dialysis be suggested formation

and carbon at carbon

of

6 of the

5 (IIa)

of

suggests

that

techniques

which

material,

might

FdUMP:thymidylate

examination

constants

equilibrium

Observation

lead

to

stoichiometries.

its

synthetase microcalorimetry

that

strength

the

pyrimidine the

structure. for

Micromolar

the

binding dichroism

or gel

of these

bond between ring.

enolate

synthetase

by circular

(14,15),

a covalent

into

of

been determined

dUMP, dTMP, and FdUMP to thymidylate

(16,17),

II.

and free

to observe

dissociation

in Table

filtration

complexes

the

catalytic

Delocalization

of

tautomer

(IIb)

could

negative

provide

-

deoxyribose-PO4

deoxyribose-PO4 e

resonance primed

stabilization for

electrophilic

b

II for

the attack

complex. at carbon

1514

This

binary

complex

5 by CH2H4folate

would to

be

generate

BIOCHEMICAL

Vol. 83, No. 4, 1978

the

covalent

shift

ternary

changes

shielding complex

free

results

As further

on the

has been

shown by the

for

the

The binary

carbon

with 6 of

the

does

not

this

possibility

trap

absence

of

in rapid

binary

complex

could

the

the

enzyme

observations

filtration and that

the

1.4

to

free

the to

ligand.

predicted anion,

IIb)

result

in a

ppm to hioher

be in a rapid

and breaking

a rate

protein.

shielding

that

of

exchange

a bond to

addition

The nmr results

comparable,

fluorine

nucleus

binds

dichroic

dUMP to the glycosyl

the syn.

together (5)

the

circular

nucleus

4 (in

than

making

linewidths

experiments

when FdUMP alone

the

are

to the

FdUMP (18)

at such

FdUMP on the the

complex,

binary

FdUMP in the

of

SDS

argue

against

presence

indicating

that

the

those

from

nitrocellulose

the

binary

complex

only

slight

and

system

is

not

exchange.

These

of

ring

since

higher

shift

free

be considered cysteine

pyrimidine

ppm to

complex.

catalytic

the

of

chemical

SDS to the

at carbon

rather

native

1.9

of

binary

oxygen

shielding,

complex

in chemical

pH dependence

to lower

to

addition

a covalent

exocyclic

RESEARCH COMMUNICATIONS

ternary

12.4

identical

against

charge

situation

However,

in a spectrum

negative

observed

of SDS from

FdUMP.

evidence

2 ppm shift

The covalent

complex.

upon addition

from

AND BIOPHYSICAL

enzyme

away from

chemical This non-integral

shift study

the

but

agrees

with

1.6

FdUMP binding

et --

with

with group

respect

to

free

of

recent sites

binding circular per

the

for

dichroism enzyme

dimer

1515

in environment

suggested

the

anti

which

by their that

binding

pyrimidine

ring

conformation

would

about

toward

move the

fluorine

lead

to a small

ligand.

confirmation sites

have

FdUMP would

phosphate

non-covalent

As indicated

of

preferred

type

further

(16)

a rotation

solution

this

al.

is

changes

synthetase.

5'

provides

number

Leary

the

of

with

that

undergoes

occurs

from

A rotation

suggest

to thymidylate

studies,

bond

with

of the

presence

FdUMP as noted studies were

found

of

in earlier

by Plese

(17)

in Tris-Cl

a studies, where

at pH 7.4.

Vol. 83, No. 4, 1978

This

BIOCHEMICAL

stoichiometry

of our

enzyme

reagents

also

corresponds

preparation

measured

to the with

RESEARCH COMMUNICATIONS

catalytic

cysteine

a variety

content

of sulfhydryl

(19,20).

Studies

are

ongoing

to

further

19F , 31P and l3 C nmr in order on formation about

AND BIOPHYSICAL

of the

enhancements

to examine

complex in the

characterize the

effect

and how various binding

of

the

binary

complex

which

folate

by

phosphate

derivatives

FdUMP to thymidylate

exerts

bring synthetase

(14).

We are grateful to William A Munroe for the preparation Acknowledgements: of FdUMP employed in these studies. Support by the American Cancer Society through a Faculty Research Award (FRA-144) to R. B. D. and the Alfred P. Sloan Foundation Fellowship to P. D. E. is greatly appreciated.

REFERENCES 1.

Friedkin,

M. (1973)

2.

Danenberg,

3.

Reyes,

4.

Langenbach,R. J., Biochem. Biophys.

5.

Santi, D. V. and McHenry, 69, 1855-1857.

6.

Santi, D. V., 471-481.

McHenry,

C. S.,

Sommer,

7.

Santi, D. V., lJ, 467-470.

McHenry,

C. S.,

and Perriard,

8.

Byrd, R. A., Dawson, W. H., Ellis, J. Amer. Chem. Sot. 99, 6139-6141.

9.

Byrd,

10.

Lyon, J. A., Pollard, A. L., Loeble, Cancer Biochem. Biophys. 1, 121-128.

11.

Wahba,

12.

Dawson, W. H., Cargill, Nucleosides, Nucleotides

13.

Byrd,

14.

Galivan, J. H., 15, 356-362.

P. V.

Advan.

(1977)

Enzymol.

Biochim.

P. and Heidelberger,

R. A.

A. J.

3&,

235-292.

Biophys.

C. (1965)

Acta

Mol.

473,

73-92,

Pharmacol.

1,

Danenberg, P. V., and Heidelberger, Res. Commun. 48, 1565-1571.

(1977)

Ph.D.

and Friedkin,

R. A. and Ellis, Maley,

C. S.

Thesis,

(1972)

Proc.

P. D.,

and Dunlap, of

G. F.,

and Maley,

1516

Biochemistry

R. B,

Chem. 236,

Res. 2fj,

F. (1976)

l3,

Carolina.

R. B. (1977)

J. Mag.

USA

R. B. (1977)

South

J. BIol.

R. L., and Dunlap, 4, 363-375.

Sci.

Biochemistry

R. B. and Dunlap,

(1961)

P. D. (1977)

Acad,

E. R, (1974)

University

M. J.

C. (1972)

Natl,

H. (1974)

14-30.

3,

(1975) PCll-PC12.

Carbohyd.,

169-173. Biochemistry

Vol. 83, No. 4, 1978

8lOCHEMlCAL

15.

Beaudette, N. V., Langermann, (1977) Arch. Biochem. Biophys.

16.

Leary, R. P., Beaudette, Chem. 250, 4864-4868.

17.

Plese,

P. C. (1978)

18.

Lewis,

C. A. Jr.,

19.

Plese,

P. C. and Dunlap,

20.

Lewis,

C. A. Jr.,

and Kisliuk,

Thesis,

unpublished

RESEARCH COMMUNICATIONS

N., Kisliuk, R. L., 179, 272-278.

N. V.,

Ph.D.

Munroe,

AND BIOPHYSICAL

R. L.

University

and GaumOnt, (1975)

of South

J.

Y. Biol.

Carolina.

results.

R. B. W. A.,

(1977)

J.

Biol.

and Dunlap,

1517

Chem. 252, R. B.,

6139-6144.

unpublished

results.

19F NMR of the 5-fluorodeoxyuridylate-thymidylate synthetase binary complex.

Vol. 83, No. 4, 1978 8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS August 29,1978 Pages "F NMR OF THE 5-FLUORODEOXYURIDYLATE-THYMIDYLATE...
403KB Sizes 0 Downloads 0 Views