A R T I C L E S
Schwartz et al.
echocystis sp DnaB intein24 followed by the hTP gene. As per the
vector protocol, hTP was subcloned in such a way as to leave no
vector derived amino acids after intein cleavage during the enzyme
purification. The hTP construct was overexpressed in the K BR2566
(T7 express) cell strain of Escherichia coli (New England Biolabs).
A typical expression is described as follows: Cells were grown in
1 L baffled flasks containing LB broth and 100 µg/mL carbenicillin
at 37 °C to an OD600 of 0.7 with shaking. After sufficient cell density
was reached, cells were placed at 4 °C for 30 min, augmented with
1 mM IPTG, and shaken at 16 °C and 150 rpm for 24 h. Cells
were harvested by centrifugation and frozen in liquid nitrogen. A
typical expression resulted in approximately 3 g of cells per 1 L of
culture. Cells were thawed and resuspended in chilled buffer
containing 100 mM HEPES pH 8.5, 1 mM EDTA, and Roche Mini-
Complete, EDTA-free protease inhibitor cocktail (1 pill/10 mL
buffer) at a ratio of 1 g of cells/4 mL of lysis buffer. During the
entire purification, the enzyme preparation was maintained at 4 °C.
Cells were lysed with 3 passes through a French press. The sample
was clarified by centrifugation and loaded onto 20 mL of chitin
affinity resin (New England Biolabs) in a 3 cm diameter column
pre-equilibrated with loading buffer containing 20 mM HEPES pH
8.5, 500 mM NaCl, and 1 mM EDTA at a flow rate of 2 mL/min.
The column was washed with 15 column volumes of loading buffer
at a rate of 4-6 mL/min. After washing, the column was
equilibrated with 10 column volumes of cleavage buffer containing
20 mM sodium phosphate pH 6.5, 500 mM NaCl, and 1 mM EDTA
at a flow rate of 6 mL/min. After equilibration, the pH of the eluent
was found to be 6.5. The column was incubated in the cold room
overnight, and the cleaved hTP was eluted at a rate of 2 mL/min.
The enzyme was detected by SDS-PAGE and was electrophoreti-
cally homogeneous. The enzyme was concentrated by ultrafiltration
to ∼20 mg/mL as determined by the calculated molar extinction
coefficient of 23,490 M-1 cm-1 at 280 nm. The concentrated
enzyme was dialyzed against 20 mM potassium phosphate buffer
at pH 7, aliquoted, and frozen in glass vials with liquid-nitrogen-
cooled isopentane. A typical expression yielded 5 mg of hTP/L of
culture with a specific activity of 10 U/mg at 22 °C for the
phosphorolysis of dT as measured by the Cary 300 spectrophoto-
metric assay (see below).
Because stock enzyme is stored in phosphate to increase stability,
a routine dialysis method was developed. Before use in any
experiment, stock enzyme was thawed on ice, inserted into a 0.5
mL dialysis cassette, and dialyzed against argon-saturated 20 mM
HEPES pH 7.4 buffer at 4 °C with 7 × 300 mL exchanges over
20 h. A constant stream of argon was bubbled through the buffer
during dialysis.
Nucleotide and Nucleoside HPLC. A three-step preparative
method was developed to purify all radiolabeled nucleosides and
nucleotides using a Waters 600C HPLC outfitted with a model 486
UV detector. Samples were monitored at 260 nm and run at a flow
rate of 1 mL/min. The first step was also used as an analytical
method for quantification of nucleoside related products.
In the first step, reaction products were separated on a 4.6 mm
× 250 mm Waters YMC ODS-A analytical C18 reversed phase
column (5 µm particle size) using a gradient of solvent A (100
mM potassium phosphate buffer pH 6.0 and 6 mM tetrabutylam-
monium bisulfate) and solvent B (100 mM potassium phosphate
buffer pH 6.0 and 6 mM tetrabutylammonium bisulfate in 30%
acetonitrile). Separation proceeded with the following elution
program: isocratic 100% A for 5 min, ramp to 30:70 A/B over 25
min, isocratic 30:70 A/B for 10 min. Samples were collected and
evaporated to a small volume for reinjection.
In the second step, reaction products were separated on a 4.6
mm × 250 mm Waters Delta Pak analytical C18 reversed phase
column (15 µm particle size) using a gradient of solvent A (water)
and solvent B (50% methanol). Separation used the elution program
Figure 1. hTP-catalyzed hydrolytic depyrimidination of dT.
transition state. For nucleophilic substitution reactions, this
allows a concerted bimolecular process (ANDN) to be distin-
guished from a stepwise process (DN*AN).16 Furthermore,
stepwise mechanisms can be resolved with respect to being rate-
‡
limiting at leaving group departure (the first step, DN *AN) or
‡
at nucleophilic attack (the second step, DN*AN ).
The physiological reaction of hTP uses inorganic phosphate
as the nucleophile, but this reaction is not amenable to KIE
approaches because of kinetic complexity obscuring the chemi-
cal step(s). The slow hydrolytic reaction permits intrinsic KIE
measurements (Figure 1). Multiple KIEs are used to demonstrate
this reaction to proceed through a stepwise DN*AN‡ mechanism.
The V/K KIEs determined for this reaction are the product of
the KIEs on the nucleophilic attack (AN) step and the equilibrium
isotope effect (EIE) from the equilibrium between the 2-deox-
yribosyl oxocarbenium ion intermediate and free dT. This
transition state differs from that reported earlier for the reaction
using AsO4 as the substrate analogue.17
Materials and Methods
3
Materials. H- and 14C-labeled riboses and glucoses and [5′-
3H]dT were purchased from American Radiolabeled Chemicals. 15N-
labeled thymine and thymidine phosphorylase inhibitor (5-chloro-
6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride, TPI)18 were
generous gifts from Industrial Research Limited (Lower Hutt, New
Zealand). Methyl 4,6-O-benzylidene-ꢀ-D-glucopyranoside (HDH
Pharma, San Diego, CA), tetrabutylammonium bisulfate (Fluka),
and 2-deoxyribose (dRib, Acros) were purchased commercially.
Ultima Gold scintillation fluid (Perkin-Elmer) was used for all
scintillation counting. Acetonitrile, methanol, trifluoroacetic acid,
and 14.6 cm glass Pasteur pipettes for charcoal columns were
purchased from Fisher. Ribonucleotide-triphosphate reductase was
a generous gift from Gary Gerfen (Albert Einstein College of
Medicine).19,20 Ribokinase,21 phospho-D-ribosyl-1-pyrophosphate
synthase,21 and adenine phosphoribosyltransferase22 were prepared
as described previously. All other reagents and synthetic enzymes
were from Sigma-Aldrich.
Preparation of hTP. The synthetic gene encoding hTP was
purchased in an expression vector from DNA 2.0. The hTP gene
was subcloned into the pTWIN1 intein expression vector (New
England Biolabs), resulting in a construct with an N-terminal chitin
binding domain23 fused to a mini-intein derived from the Syn-
(16) Guthrie, R. D.; Jencks, W. P. Acc. Chem. Res. 1989, 22, 343–349.
(17) Birck, M. R.; Schramm, V. L. J. Am. Chem. Soc. 2004, 126, 2447–
2453.
(18) Matsushita, S.; Nitanda, T.; Furukawa, T.; Sumizawa, T.; Tani, A.;
Nishimoto, K.; Akiba, S.; Miyadera, K.; Fukushima, M.; Yamada,
Y.; Yoshida, H.; Kanzaki, T.; Akiyama, S. Cancer Res. 1999, 59,
1911–1916.
(19) Blakley, R. L. Methods Enzymol. 1978, 51, 246–259.
(20) Booker, S.; Stubbe, J. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8352–
8356.
(21) Merkler, D. J.; Kline, P. C.; Weiss, P.; Schramm, V. L. Biochemistry
1993, 32, 12993–13001.
(22) Shi, W.; Tanaka, K. S.; Crother, T. R.; Taylor, M. W.; Almo, S. C.;
Schramm, V. L. Biochemistry 2001, 40, 10800–10809.
(23) Chong, S.; Mersha, F. B.; Comb, D. G.; Scott, M. E.; Landry, D.;
Vence, L. M.; Perler, F. B.; Benner, J.; Kucera, R. B.; Hirvonen, C. A.;
Pelletier, J. J.; Paulus, H.; Xu, M. Q. Gene 1997, 192, 271–281.
(24) Wu, H.; Xu, M. Q.; Liu, X. Q. Biochim. Biophys. Acta 1998, 1387,
422–432.
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13426 J. AM. CHEM. SOC. VOL. 132, NO. 38, 2010