Weak binding affinity of human 4EHP for mRNA
JOANNA ZUBEREK,1,4DOROTA KUBACKA,1,4AGNIESZKA JABLONOWSKA,2JACEK JEMIELITY,1
JANUSZ STEPINSKI,1NAHUM SONENBERG,3and EDWARD DARZYNKIEWICZ1
1Department of Biophysics, Institute of Experimental Physics, Warsaw University, 02-089 Warsaw, Poland
2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
3Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, H3G 1Y6, Canada
Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E,
with the mRNA 59 cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which
helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not
eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset
mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs
using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m7GpppG
and m7GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure
of most mRNAs.
Keywords: 4EHP; eIF4E isoforms; mRNA 59 cap; binding affinity; stopped-flow
All nuclear transcribed eukaryotic mRNAs possess a com-
mon structure called a ‘‘cap’’ at their 59 end, which consists
of 7-methylguanosine bound by a 59-59-triphosphate
bridge to the first transcribed nucleotide. The cap structure
is important for stabilizing the mRNA (Furuichi and
Shatkin 2000), facilitating the splicing of pre-mRNAs
(Konarska et al. 1984), promoting mRNA transport to
cytoplasm (Izaurralde et al. 1992), and facilitating the
binding of ribosomes to the mRNA (Mathews et al. 2000;
Pestova et al. 2007). Cap-dependent translation begins with
recognition of the cap structure by eIF4E, which forms a
heterotrimeric complex with eIF4A, which is thought to
melt the mRNA 59 secondary structure and the scaffolding
protein eIF4G that binds other factors to recruit the
ribosome (Mathews et al. 2000). The interaction of eIF4E
with eIF4G is controlled by a group of proteins generally
known as eIF4E inhibitory proteins, which share a common
eIF4E-binding site with eIF4G. Whereas some 4EBPs
repress translation of a large number of mRNAs by asso-
ciating only with eIF4E (Raught et al. 2000), others, more
recently discovered, such as Cup or Maskin, inhibit trans-
lation of specific mRNAs by binding simultaneously to
eIF4E and additional proteins that interact with sequence
elements in the mRNA 39 UTR (Wilhelm et al. 2003; Cao
and Richter 2002).
Crystallographic, NMR, and biophysical studies deter-
mined the amino acids in eIF4E that are of primary impor-
tance for cap structure recognition (Marcotrigiano et al.
1997; Matsuo et al. 1997; Niedzwiecka et al. 2002; Tomoo
et al. 2003). The structural basis for the specificity of the
eIF4E–cap interaction is the sandwiching of the 7-methyl-
guanine base via p–p stacking interactions between tryp-
tophan indol rings (Trp56 and Trp102). This interaction
is stabilized by van der Waals contacts of the m7G with
Trp166 and hydrogen bonds with Glu103. The phosphate
chain of the cap structure forms direct or water-mediated
hydrogen bonds with NH groups of Trp102 and Trp166 as
well as with side chains of lysine and arginine residues
(Arg112, Arg157, and Lys162).
eIF4E homologs have been identified in most organisms
(except for Saccharomyces cervisiae). In mammals, there are
three members of the eIF4E family: eIF4E (eIF4E-1), 4EHP
4These authors contributed equally to this work.
Reprint requests: Edward Darzynkiewicz, Department of Biophysics,
Institute of Experimental Physics, Warsaw University, Zwirki i Wigury 93,
02-089 Warsaw, Poland; e-mail: email@example.com; fax: +48 22 55 40771.
Article published online ahead of print. Article and publication date are
RNA (2007), 13:691–697. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2007 RNA Society.
(eIF4E-2), and eIF4E-3 (Rom et al. 1998; Joshi et al. 2004).
Based on sequence alignments, h4EHP, together with nCBP
from plants (Ruud et al. 1998), IF4E-4 from Caenorbiditis
elegans and d4EHP (deIF4E-8) from Drosophila mela-
nogaster are members of the eIF4E-2 class (Joshi et al.
2005). Members of this class possess mainly Tyr/Phe in the
position corresponding to Trp43 and Tyr in the position of
Trp56 of human eIF4E (Joshi et al. 2005). In h4EHP both
of these positions are occupied by tyrosine (Fig. 1). Mouse
and human 4EHP binds to m7GTP-Sepharose but does not
interact with eIF4G (Rom et al. 1998; Joshi et al. 2004) and
cannot rescue the lethality of eIF4E gene deletion in yeast
(Joshi et al. 2004). Recently, the Drosophila 4EHP has been
reported to be a translation repressor that is required for
the asymmetric distribution of caudal protein in the egg,
which is essential for the appropriate development of the
embryo (Cho et al. 2005). d4EHP blocks translation by
binding to the 59 cap structure of Caudal mRNA and to the
regulatory protein Bicoid, which interacts with 39 UTR of
the caudal mRNA (Cho et al. 2005).
Here we present data on the binding affinities of cap
analogs for human 4EHP versus human eIF4E using the
fluorescence titration method and stopped-flow technique.
RESULTS AND DISCUSSION
All proteins were expressed in Escherichia coli in inclusion
bodies, refolded by one-step dialysis, and purified using
ion-exchange chromatography to avoid any contact with
cap analogs (Niedzwiecka et al. 2002). Analysis of cell
extracts and proteins after dialysis showed limited pro-
teolysis of the N terminus. However, the application of
ion-exchange column resulted in homogeneous full-length
proteins (>95%; Fig. 2). The amount of proper refolded
protein, obtained from fluorescence titrations by fitting
concentration of the ‘‘active’’ protein as a free parameter of
the equilibrium equation, was about 70%–85% (Niedzwiecka
et al. 2002).
Different binding affinities of 4EHP and eIF4E
for cap analogs
The interaction between cap analogs and eIF4E results in
quenching of intrinsic Trp fluorescence (Fig. 3A). 4EHP
possesses six out of the eight conserved tryptophan residues
of eIF4E and an additional tryptophan in the C terminus.
Trp43 and Trp56 of eIF4E are replaced in 4EHP by
tyrosines (Rom et al. 1998; Joshi et al. 2004). These
differences result in slightly lower fluorescence intensities
for equimolar protein solutions of 4EHP versus eIF4E.
However, the emission spectra of both proteins are very
similar (data not shown), suggesting no significant struc-
tural differences between the proteins as a result of the
changes in the environment of Trp residues.
To determine the accurate association constants (Kas) for
the complexes of 4EHP with a series of mono- and dinu-
cleotide cap analogs, we applied the time-synchronized
fluorescence titration method (Niedzwiecka et al. 2002)
and the stopped-flow technique with an emission detector
(Dlugosz et al. 2002). The Kas values obtained from
fluorescence titration are shown in Table 1, and the
corresponding Gibbs free energy of binding (DG°) are
graphically presented in Figure 3B. The
kinetic parameters for the association
reaction protein–cap analog are shown
in Table 2. As a control, Kasfor com-
plexes of human eIF4E with all tested
cap analogs were determined. It is
striking that 4EHP binds all cap analogs
significantly weaker than eIF4E. How-
ever, the differences among binding
affinities depend on the nature of the
cap analog. The association constant for
4EHP with m7GTP is about 100-fold
lower compared to that for eIF4E
(Fig. 3A), whereas the dinucleotide
triphosphate cap analogs (m7GpppG,
m7GpppA, and m7GpppC) are bound
by 4EHP about 30-fold weaker. These
data were also confirmed by stopped-
flow measurements (Table 2). The
results are in good agreement with the
observations that human and Drosoph-
ila 4EHP bind m7GTP-Sepharose less
efficiently than their eIF4E counterparts
FIGURE 1. Amino acid alignment of human eIF4E (gi:4503535) with 4EHP (gi:3172339)
performed with CLUSTALW (Thompson et al. 1994). Residues that are identical in proteins
are shadowed in black and conserved substitutions in gray. The conserved residues that play a
role in cap binding by eIF4E (Marcotrigiano et al. 1997), with changes in 4EHP, are labeled
above. Stars below the lines indicate the positions of eight evolutionarily conserved tryptophan
residues in eIF4E.
Zuberek et al.
RNA, Vol. 13, No. 5
(Tee et al. 2004; Cho et al. 2005). However, the differences
of two orders of magnitude in binding affinities for the cap
structure between the two proteins are striking, particularly
because 4EHP, compared to eIF4E, possesses only three
substitutions in the cap-binding slot: two conservative and
one nonconservative (Fig. 1).
Replacement of Trp56 by Tyr in eIF4E moderately
stimulates cap binding affinity
A mutant of human eIF4E possessing a leucine substitution
of Trp56 fails to bind m7GTP-Sepharose (Morino et al.
1996). However, substitution of the corresponding trypto-
phan by phenylalanine in yeast eIF4E reduces eIF4E’s
binding to the cap structure by 50% only (Altmann et al.
1988). To elucidate the contribution of the 4EHP tyrosine
residue (corresponding to Trp56 in eIF4E) to the weak
binding affinity of 4EHP for the cap, we generated a mutant
of eIF4E possessing tyrosine instead of tryptophan in
position 56. Surprisingly, this mutant exhibits a slightly
enhanced (z1.5-fold) binding to the cap structure (Table 1).
The Kasfor the complex of mutated eIF4E with m7GTP is
109.7 6 5.0 mM?1, whereas for wild type eIF4E is 68.41 6
5.09 mM?1. The increase of Kasfor eIF4EW56Y is very
similar for all tested cap analogs and does not depend on
the number of phosphate groups. These results show that in
eIF4E two tryptophans as well as tryptophan and tyrosine
residues can sandwich 7-methylguanine through the p–p
stacking interaction with similar efficiency. The recognition
mode of the cap structure by the sandwich stacking of the
m7G moiety via two aromatic rings of amino acids is
exercised by other, eIF4E-unrelated, mRNA 59 cap binding
proteins. The vaccinia virus mRNA-cap-dependent 29-O-
methyltransferase, VP39, contains a tyrosine and a phenyl-
alanine (Tyr22 and Phe180) (Hu et al. 1999) and the
conserved nuclear cap binding protein, CBC20, contains
two tyrosines (Tyr20 and Tyr43) (Mazza et al. 2002). The
crystallographic structures of cap binding proteins sup-
ported by theoretical studies showed that the almost perfect
alignment of the aromatic rings, an interplanar distance of
3.1–3.6 A˚, a substantial area of overlap in stacking rings,
and the positive charge of m7G all contribute to the strong
interaction between the p-electrons of stacked rings
(Quiocho et al. 2000; Fechter and Brownlee 2005). The
VP39, which possesses phenylalanine in the stacking mode,
had the lowest binding affinity (Kasz0.1 mM?1), and its
replacement by tryptophan or tyrosine enhances binding.
On the other hand, substitution of phenylalanine for Tyr22
cannot rescue the stacking interaction (Hu et al. 1999;
Hsu et al. 2000). The CBC20, in which two tyrosines are
involved in stacking, exhibits binding affinity for m7GTP
similar to eIF4E: Kas= 29.7 6 2.3 mM?1for CBC (Worch
et al. 2005) and Kas= 33.28 6 1.04 mM?1for mouse eIF4E
in buffer containing 200 mM KCl (Zuberek et al. 2004).
The ring system of tyrosine is more p-electron rich than
that of phenylalanine due to the electron-donating charac-
ter of phenolic oxygen, which engenders a stronger stacking
interaction between tyrosine and the positively charged
m7G base. Thus, all these data suggest that the presence of
Tyr78 instead of tryptophan cannot be the major reason for
the 100-fold weaker binding of cap analogs to 4EHP than
to its eIF4E counterpart. In human eIF4E Trp56 is located
in loop S1–S2 between two b sheets (Tomoo et al. 2003).
This region in 4EHP contains five additional amino acids
(Fig. 1), which can increase the flexibility of this loop. As a
consequence, it may prevent the correct parallel alignment
of the aromatic ring of Tyr78 and its substantial overlap
with the m7G moiety essential for an efficient stacking
interaction, which is adopted in the eIF4EW56Y mutant.
Recently, the cap binding affinities of four Leishmania
eIF4E isoforms were determined (Yoffe et al. 2006). They
all possess binding affinities for m7GTP similar to h4EHP.
However, in LeishIF4E-1 two tryptophan residues and in
LeishIF4E-4 one tyrosine and one tryptophan are involved
in the stacking interaction. LeishIF4E-4 binds m7GTP even
approximately fourfold stronger than LeishIF4E-1, which is
in agreement with our results for the eIF4EW56Y mutant.
These data suggest that not only is the type of aromatic
amino acid (Tyr or Trp) engaged in the stacking interaction
important for stabilizing the complex of the eIF4E-cap, but
other structural elements also are important. However,
homology modeling of either Leishmania isoforms (Yoffe
et al. 2006) or h4EHP (Rom et al. 1998) did not predict
significant differences in the three-dimensional structure
compared to eIF4E.
Effect of the negative charge of cap analogs on their
affinity for 4EHP
It has been shown previously that electrostatic interactions
play a crucial role in the recognition of the cap structure by
eIF4E (Niedzwiecka et al. 2002; Zuberek et al. 2004). The
extension of the phosphate chain in cap analogs results in
systematic, marked enhancement of the binding affinity for
FIGURE 2. Purified on ion-exchange column proteins visualized on
a Comassie-stained 15% SDS-PAGE gel. Lane 1, protein marker
weight standards (Sigma); lane 2, human eIF4E; lane 3, human
eIF4EW56Y; and lane 4, human 4EHP.
Weaker binding of mRNA cap to 4EHP than to eIF4E
eIF4E. 4EHP carries only two substitutions, with reference
to positive charged amino acid in the cap binding slot of
eIF4E (Fig. 1). A hydrophobic isoleucine substitutes for
Lys162, which forms a direct hydrogen bond with an
oxygen atom of the b-phosphate group (Tomoo et al.
2003). In addition, a lysine replaces Arg112, which in eIF4E
interacts with the a-phosphate group by a water-mediated
hydrogen bond (Marcotrigiano et al. 1997; Tomoo et al.
2003). To establish the possible effect of
substitutions on cap binding (especially
the lack of a lysine residue), we analyzed
the Gibbs free energy of binding in
relation to the number of phosphate
groups within the cap analogs (Fig. 3B).
The addition of the b-phosphate group
to the cap analog (m7GMP/m7GDP)
gives a change in binding free energy
of about ?0.7 kcal/mol for 4EHP and
about ?1.8 kcal/mol for eIF4E. The
difference between proteins of about
1.1 kcal/mol in the energetic cost
(DDGo) corresponds to one hydrogen
bond. For both proteins the addition
(m7GDP/m7GTP) causes a compara-
ble change of binding free energy
(4EHP of about ?0.65 kcal/mol; eIF4E
of about ?0.8 kcal/mol). This suggests
that the lack of the lysine residue in
4EHP is partially responsible for the
z100-fold decrease in its binding affin-
ity for m7GDP and m7GTP compared
to eIF4E, whereas for m7GMP it is only
10-fold. The weaker binding of m7GMP
by 4EHP presumably results from the
replacement of arginine by lysine with a
shorter side chain, thus preventing the
formation of a water-mediated hydro-
gen bond. The twofold increase in the
association constant for m7Gp4(Kas=
1.20 6 0.04 mM?1) in comparison with
m7GTP (Kas = 0.70 6 0.04 mM?1)
suggests that the d-phosphate of the
cap analog in contrast to eIF4E (three-
fold increase of Kas) does not interact
directly with the protein by forming
Earlier studies suggested that h4EHP
and d4EHP bind a cap structure weaker
than eIF4E based on qualitative mea-
(Tee et al. 2004; Cho et al. 2005). Here
we used quantitative measurements to show that 4EHP
binds to the cap structure with a very low affinity (30–100-
fold lower than eIF4E). The weak binding of 4EHP to the
cap prevents it from competing with eIF4E for association
with the mRNA cap and to inhibit translation. Translational
inhibition of caudal mRNA occurs only when 4EHP
simultaneously binds the cap and Bicoid. The association
of d4EHP with Bicoid might enhance its affinity for the cap.
FIGURE 3. (A) Fluorescence titration curves for binding m7GTP to human eIF4E (D), its
eIF4EW56Y mutant (d), human 4EHP (j), and fitting residuals. Titrations were carried out in
50 mM HEPES/KOH (pH 7.2), 0.5 mM EDTA, and 1 mM DTT adjusting to I = 150 mM by
KCl at 20°C. Protein fluorescence, presented as relative value, was excited at 280 nm and
observed at 337 nm. The observed increasing fluorescence signal at a higher concentration of
m7GTP originates from free-cap analog emission. (B) Graphical comparison of Gibbs free
energy of binding (DGo) for association of cap analogs with human eIF4E, its mutant, and
human 4EHP, calculated from obtained association constants (Kas).
Zuberek et al.
RNA, Vol. 13, No. 5
A similar mechanism of translational inhibition by h4EHP
may occur by binding to as yet unidentified proteins.
MATERIALS AND METHODS
Synthesis of cap analogs
Syntheses of mono- and dinucleotide cap analogs were performed
as described previously (Darzynkiewicz et al. 1985, 1990; Jemielity
et al. 2003; Zuberek et al. 2004). The cap analog concentrations
were determined spectrophotometrically (Cai et al. 1999).
Cloning and mutagenesis
The cDNA of human 4EHP, amplified from the pCDNA3-
HA_4EHP vector by PCR, was subcloned into the expression
vector pET30a (Novagen) in NdeI–BamHI sites.
The QuikChange PCR-based site-directed mutagenesis kit
(Stratagene) was used to obtain the point-mutations replacement
of Trp56 by tyrosine in human eIF4E. As an initial template the
cDNA for eIF4E in a pET11d vector (Novagen) was used and site-
directed mutagenesis was performed exactly according to the
instructions provided by Stratagene. The presence of the mutation
was confirmed by automatic DNA sequencing.
Protein expression and purification
For expression of the human eIF4E, an eIF4EW56Y mutant and
4EHP appropriate plasmids were transformed into E. coli
BL21(DE3) cells. Bacteria were grown in LB
medium to OD600 nmof 0.8, and induced for
3 h at 37°C by adding 0.5 mM isopro-
Cells were harvested, resuspended in lysis
buffer (20 mM HEPES/KOH [pH 7.5], 100
mM KCl, 1 mM EDTA, 2 mM DTT, and
10% glycerol) and disrupted by sonication.
After centrifugation of the lysate (30,000g for
30 min) the supernatant was removed and the
pellet was washed three times with wash
buffer (20 mM HEPES/KOH [pH 7.2], 1 M
guanidine hydrochloride, 2 mM DTT, and
10% glycerol). The inclusion bodies were
dissolved in 50 mM HEPES/KOH (pH 7.2),
6 M guanidine hydrochloride, 10% glycerol,
and 2 mM DTT, and cell debris was removed
by centrifugation (43,000g for 30 min). The
protein (diluted to a concentration lower
than 0.1 mg/mL) was refolded by one-step dialysis against 50 mM
HEPES/KOH (pH 7.2), 100 mM KCl, 1.0 mM EDTA, and 2 mM
DTT, and purified by ion exchange chromatography on a HiTrap
MonoSP column (Amersham Bioscience). The purified proteins
were analyzed by SDS-PAGE and their concentrations were deter-
mined by absorption, assuming the following: e280 = 53,400
M?1cm?1for eIF4E, e280= 49,200 M?1cm?1for eIF4EW56Y,
and e280= 47,600 M?1cm?1for 4EHP (calculated from amino
acid composition using an algorithm on the ExPASy Server).
Fluorescence binding titration
Fluorescence titration measurements were carried out on an
LS-50B or LS-55 spectrofluorometer (Perkin-Elmer), in 50 mM
HEPES/KOH (pH 7.2) and 0.5 mM EDTA 1 mM DTT, adjusting
to an ionic strength of 150 mM by KCl at 20.0 6 0.2°C. Aliquots
of 1 mL increasing concentrations of cap analog solutions were
added to 1.4 mL of 0.1 or 0.2 mM protein solutions. Fluorescence
intensities (excitation at 280 nm or 295 nm with a 2.5-nm bandwidth
and detection at 337 nm or 345 nm with a 4-nm bandwidth and a
290-nm cutoff filter) were corrected taking into account sample
dilution and the inner filter effect. Equilibrium association constants
(Kas) were determined by fitting the theoretical dependence of the
fluorescence intensity on the total concentration of the cap analog
to the experimental data points according to the equation described
previously (Niedzwiecka et al. 2002). The concentration of protein
was fitted as a free parameter of the equilibrium equation showing
the amount of ‘‘active’’ protein. The final Kaswas calculated as a
weighted average of 3–10 independent titrations, with the weights
taken as the reciprocals of the numerical standard deviations
TABLE 1. Equilibrium association constants (Kas) for the complexes of cap analogs with
the human eIF4E, eIF4EW56Y mutant, and human 4EHP obtained from analysis of
steady-state fluorescence titrations at 20°C
0.78 6 0.04
17.76 6 0.34
68.41 6 5.09
221.5 6 12.9
5.94 6 0.39
6.13 6 0.34
3.97 6 0.21
4.76 6 0.19
47.88 6 2.12
0.027 6 0.001
1.79 6 0.04
29.5 6 0.6
109.7 6 5.0
272.6 6 10.6
7.00 6 0.12
7.42 6 0.16
4.33 6 0.08
5.09 6 0.14
69.4 6 2.3
0.051 6 0.002
0.07 6 0.01
0.23 6 0.03
0.70 6 0.04
1.20 6 0.04
0.17 6 0.01
0.12 6 0.01
0.13 6 0.01
0.16 6 0.02
0.72 6 0.02
0.031 6 0.002
TABLE 2. Rate constants for association (k+1) and dissociation (k?1) of human eIF4E and 4EHP with cap analogs obtained from fitting
one-step model to the kinetic traces registered during stopped-flow experiments at 20°C
690.9 6 7.9
163.1 6 2.7
10.2 6 0.2
35.3 6 0.4
67.7 6 1.8
4.6 6 0.1
77.7 6 13.9
35.9 6 25.1
105.1 6 3.4
154.0 6 7.2
0.73 6 0.13
0.23 6 0.16
Weaker binding of mRNA cap to 4EHP than to eIF4E
squared. Numerical nonlinear least-squares regression analysis was
performed using ORGIN 6.0 (Microcal Software).
The Gibbs free energy of binding was calculated from the Kas
value according to the standard equation DG° = ?RTlnKas.
Stopped-flow measurements and analysis of kinetics
Kinetic measurements of interaction eIF4E and 4EHP proteins
with m7GTP and m7GpppG were run on a SX.18MV stopped-flow
reaction analyzer (Applied Photophysics) using fluorescence
detection. The protein emission was excited at 290 nm (with a
0.5 mm slit) and its fluorescence was monitored after passage
through a 320-nm cutoff filter. The path lengths in the stopped
flow cell were 2 mm for absorption and 10 mm for emission. The
reaction was initiated by mixing an equal volume of protein
solution (1 mM) with the cap analog (0.5–8 mM for eIF4E and
0.5–20 mM for 4EHP). The measurements were performed in
50 mM HEPES/KOH (pH 7.2), 0.5 mM EDTA adjusted to an
ionic strength of 150 mM by KCl at 20.0 6 0.1°C. The fluores-
cence changes were monitored up to a 200-msec recording of
1000 data points using the oversampling option of the instrument.
The kinetic traces for each concentration of the cap analog are
an average of 14 independent runs.
The obtained kinetics traces were subjected to nonlinear least-
squares regression assuming a one-step model for the protein–cap
described by differential equations for rates of changes of
concentration of molecular species
= k+1½P?½L? ? k?1½PL?
and its specific contributions to the monitored fluorescence signal
F = ½P? ? fP+½PL? ? fPL+½L? ? fL
where k+1and k?1are association and dissociation rate constants,
respectively; [P], [L], and [PL] are concentrations, and fP,fL,and
fPL, are molar fluorescence of free protein, free ligand, and
complex. Numerical analysis was performed using the DynaFit
program (BioKin) created by Peter Kuzmic (Kuzmic 1996).
We thank Dr. Jan M. Antosiewicz (Warsaw University) for
making the SX.18MV stopped-flow reaction analyzer available
to us. This work was supported by Howard Hughes Medical
Institute Grant No. 55005604 to E.D., Polish Ministry of Science
and Higher Education Grant No. 2P04A 006 28 to E.D., and the
Canadian Institute of Health Research to N.S.
Received December 30, 2006; accepted February 6, 2007.
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