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.
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Biophys.
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73-92,
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Danenberg, P. V., and Heidelberger, Res. Commun. 48, 1565-1571.
(1977)
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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
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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
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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.