Journal of the American Chemical Society
Article
acid followed by a Finnigan LCQ Deca mass spectrometer. HRMS
analysis was done on a Waters Q-TOF mass spectrometer.
Purification Methods. The trapped intermediate and 5-
hydroxymethyl-dUMP were purified by HPLC using analytical
ASSOCIATED CONTENT
Supporting Information
■
*
S
(
Discovery series 250 mm × 4.6 mm) and semipreparative (Discovery
series 250 mm × 10 mm) reversed-phase Supelco columns,
respectively. The mobile phase used for separation was a gradient of
AUTHOR INFORMATION
5
0 mM KH PO at pH 6 (for purification of the intermediate) or 100
2 4
mM KH PO4 at pH 2 (for purification of synthesized 5-
2
hydroxymethyl-dUMP) and methanol. Elution of the species of
interest was followed by UV absorbance at 267 nm. Eluent containing
the purified species was collected, lyophilized to dryness, and dissolved
Author Contributions
These authors contributed equally.
∥
in H O for LC−MS, MS−MS, and HRMS analyses.
2
Notes
Acid Quenching of FDTS during the Oxidative Half-
Reaction. A solution of oxidized FDTS (100 μM) was made
anaerobic in a sealed tonometer by cycles of applied vacuum and
equilibration with purified argon. The anaerobic enzyme was reduced
stoichiometrically with a dithionite solution, as followed spectrophoto-
metrically at 454 nm. The reduced FDTS was then mixed with dUMP
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH R01 GM065368 and NSF
CHE 0715448 to A.K., GM61087 to B.A.P., GM08270 to
J.A.C., U54 GM094586 to S.A.L., the Iowa CBB to T.V.M. and
E.M.K., and NSF GRFP to E.M.K.
(
92 μM) from a side arm of the tonometer and loaded on a KinTek
Chemical Quench-Flow instrument (model RQF-3), which had
previously been scrubbed of oxygen with a glucose/glucose oxidase
solution (50 units/mL). An anaerobic 400 μM CH H folate solution
2
4
containing 50 units/mL glucose oxidase, 10 mM glucose (to ensure
anaerobic conditions), and 30 mM formaldehyde (to stabilize
CH H folate) was prepared. FDTS reactions were initiated by rapid
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14
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2
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14
[
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μM CH H folate, and limiting [2- C]dUMP (10 μM) were manually
2
4
quenched with 1 M HCl at 1−2 s. The aliquots from both experiments
were dried by speed-vacuum and resuspended in water or neutralized
(
1
02.
(
for HCl samples) and analyzed by HPLC with a radioactivity flow
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c):
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2
k
k
2
1
dUMP → Intermediate → dTMP
(
2
(
The following set of rate equations was used in the fitting:
oxidative half-reaction in which FADH reacts to form FAD.
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2
d
dt
d
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d
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[
[
[
dUMP] = − k [dUMP]
1
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Intermediate] = k [dUMP] − k [Intermediate]
1
2
(
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dTMP] = k [Intermediate]
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dx.doi.org/10.1021/ja2120822 | J. Am. Chem. Soc. 2012, 134, 4442−4448