C O MMU N I C A T I O N S
are hypochromic, the extinction coefficients had to be measured
on the basis of the amounts of products formed by the action of
wild-type Fhit on the molecules synthesized using the engineered
H96G-Fhit.8
AMP-Im is an activated form of AMP that can be used in the
nonenzymatic synthesis of the phosphoanhydride linkage, as well
as the phosphodiester linkage.9
-13
Thus, nonenzymatic reactions
of nucleoside-5-imidazolides with nucleoside di- and triphosphates
should produce dinucleoside tri- and tetraphosphates. The nonen-
zymatic reactions are less specific and far slower than the process
with the engineered enzyme. The advantages of the engineered
enzyme can be appreciated by comparing the kinetic course of the
enzymatic and nonenzymatic production of Ap U under otherwise
3
identical conditions. Data are shown in Figure 2. The engineered
enzymatic rate is much faster than the nonenzymatic rate at all
concentrations of MgUDP and approaches saturation with a K
m
value of 20 mM for MgUDP.
Figure 1. Effect of MgUDP on the H96G-Fhit-catalyzed reactions of AMP-
Im. Initial rates of product formation as functions of increasing MgUDP
concentrations are plotted for the reaction of AMP-Im catalyzed by H96G-
Fhit. The reaction mixtures contained 100 µM MOPS buffer (pH 7.5), 2
µM H96G-Fhit, 300 µM AMP-Im (saturating), and MgADP varied from
Acknowledgment. This work was supported by Grant No.
GM30480 from the National Institute of General Medical Sciences,
U.S. Public Health Service.
Supporting Information Available: NMR spectral assignments
and MS characterizations of Ap
0
.5 to 20 mM. The reactions were quenched after 4 min by adjustment to
3
A, Ap
3 3 3 3 3
C, Ap G, Ap T, Ap U, Cp U,
pH 12 with 5 M NaOH. Aliquots of the reaction mixtures (80 µL) were
analyzed in a Beckman HPLC system equipped with an anion exchange
Tp U, dAp U, Ap A, Ap U, and Ap
3
3
4
4
4
etheno-C. This material is available
7
column, with elution and detection as described. The initial rates of Ap3A
free of charge via the Internet at http://pubs.acs.org.
formation, AMP produced, and AMP-Im disappearance were calculated.
Symbols: filled triangles, initial rate for AMP-Im disappearance; open
diamonds, initial rate for AMP production; filled diamonds, initial rate for
Ap3U formation.
References
(
1) Brenner, C. Biochemistry 2002, 41, 9003-9014.
(
2) Barnes, L. D.; Garrison, P. N.; Siprashvili, Z.; Guranowski, A.; Robinson,
A. K.; Ingram, S. W.; Croce, C. M.; Ohta, M.; Huebner K. Biochemistry
1996, 35, 11529-11535.
(
(
(
(
(
3) Huang, K.; Arabshahi, A.; Wei, Y.; Frey, P. A. Biochemistry 2004, 43,
7637-7642.
4) Lima, C. D.; D’Amico, K. L.; Naday, I.; Rosenbaum, G.; Westbrook, E.
M.; Hendrickson W. A. Structure 1997, 5, 763-774.
5) Abend, A.; Garrison, P. N.; Barnes, L. D.; Frey, P. A. Biochemistry 1999,
38, 3668-3676.
6) The chemical rescue of H96G-Fhit by imidazole requires high concentra-
3
tions, e.g. 0.5 M, and is very slow.
3
7) H96G-Fhit and AMP-Im were prepared as described. To determine the
pH optimum for Ap
3
U synthesis, the reaction mixtures consisted of 100
mM UDP, 100 mM MgCl
2
, 600 µM AMP-Im, 60 µM H96G-Fhit-H96G,
and 0.2 M MES or HEPES buffer. After reaction for 2 min at room
temperature, 40 µL samples were analyzed by HPLC, using an anion
exchange column eluted with a gradient changing from 100% buffer A
(
50 mM NaH
2 4
PO at pH 4.0) to 65% buffer B (1 M NaCl in buffer A)
-1
within 20 min at a flow rate of 2 mL min . The concentration of Ap
3
U
-1
-1 8
was calculated by using the extinction coefficient ꢀ260 ) 23.6 mM cm
.
This method was employed to obtain the transformation yields in Table
. Large-scale synthesis was conducted as follows. To 5 mL of 200 mM
MES buffer (pH 5.5) containing 80 mM UDP, 160 mM MgCl , and 0.5
1
2
mM H96G-Fhit was added solid AMP-Im to 80 mM. The reaction
proceeded to completion at room temperature in 60 min. The reaction
mixture was diluted 5-fold with double-distilled water and applied to a
pre-packed anion exchange column (200 mL of Q-Sepharose fast flow).
The column was eluted with a 2.0 L linear gradient of buffer A (10 mM
Figure 2. Nonenzymatic and H9G-Fhit-catalyzed rates of production of
Ap3U. The rates of enzymatic and nonenzymatic production of Ap3U from
AMP-Im and MgUDP were measured in a 500 µL reaction mixture
containing 200 mM MES buffer at pH 5.5, saturating AMP-Im (400 µM),
CH
rate of 2 mL min . The fractions containing Ap
to pH 7.0, and freeze-dried. The dried product was desalted by passage
through a column of Biogel P2. The yield of Ap U based on AMP-Im is
54% with 12% Ap A as a byproduct.
(8) Extinction coefficients of dinucleoside tri- and tetraphosphates at 260 nm
were determined as follows for the example of Ap U. A sample of Ap
200 µL) with a known value of absorbance at 260 nm and in 200 mM
MES buffer (pH 7.0) was completely hydrolyzed by addition of 0.01 µM
wild-type Fhit and 1.0 mM MgCl to give AMP, UDP, UMP, and ADP.
3
COONa-CH-
3
COOH, pH 4.0) to 0.5 M NaCl in buffer A at a flow
1
3
U were pooled, adjusted
6
µM H96G-Fhit, and MgUDP at 2, 5, 10, 20, and 40 mM. After 4 min at
3
room temperature, the reactions were quenched by adjustment to pH 12
with 5 M NaOH. An aliquot (40 µL) of each reaction mixture was analyzed
by HPLC and the initial rate of Ap3U formation calculated. The symbols
are as follows: triangles, initial rate for the H96G-Fhit-catalyzed reaction
2
3
3
U
(
(
the solid line is calculated from the kinetic parameters kcat ) 9.2 ( 0.7
2
-
1
min and Km for MgUDP ) 20 ( 3 mM); diamonds, initial rates for the
nonenzymatic reaction under the same conditions (the solid line represents
the fit of the second-order rate equation to the data, with k ) 52 ( 3 M
A 40 µL sample of the product mixture was analyzed by HPLC for its
content of AMP + ADP, determined spectrophotometrically using the
known extinction coefficient for these molecules (15.0 mM- cm ). The
1
-1
-
1
3
sum of AMP and ADP was taken as the total amount of Ap U, and the
-
1
min ).
in reactions with AMP-Im, and ATP does not react well. However,
at twice the concentration of MgCl employed in the synthesis of
dinucleoside triphosphates, H96G-Fhit accepts either ATP or UTP
in reaction with AMP-Im to produce Ap A or Ap U in good yields
Table 1).
Also included in Table 1 are the extinction coefficients for the
dinucleoside polyphosphates synthesized. Because these molecules
extinction coefficient for Ap3U was calculated from the measured
absorbance of the original solution. For samples that did not contain
adenosine, the same procedure was followed using the extinction
coefficient for uridine or thymidine nucleotides (10 mM- cm ).
1
-1
2
(
9) Shimazu, M.; Shiosuka, K.; Sawai, H. Tetrahedron Lett. 1990, 31, 235-238.
10) Ertem, G.; Ferris, J. P.; Sawai, K.; Hojo, T. Bull. Chem. Soc. Jpn. 1989,
62, 2018-2023.
(
4
4
(
(
11) Lesiak, K.; Torrence, P. F. J. Med. Chem. 1986, 29, 1015-102.
12) Imai, J.; Torrence, P. F. J. Org. Chem. 1985, 50, 1418-1426.
(
(13) Ertem, G.; Ferris, J. P. Origins Life EVol. Biosphere 2000, 30, 411-422.
JA0400640
J. AM. CHEM. SOC.
9
VOL. 126, NO. 31, 2004 9549
Huang, Kaisheng
