From the Indian Institute of Chemical Biology, Jadavpur, Calcutta, India 700032
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ABSTRACT |
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In the previous paper we demonstrated that
uridine-5'- UDP-glucose-4-epimerase (EC 5.1.3.2, henceforth called epimerase)
catalyzes a freely reversible reaction between UDP-glucose and
UDP-galactose. This enzyme of the galactose metabolic pathway is
essential for the biosynthesis of numerous galactoconjugates in all
cell types studied so far. This epimerase has emerged as the prototype
of a new class of oxidoreductases in which the coenzyme NAD,
noncovalently but firmly bound to the apoenzyme, acts as a true
cofactor and not as a cosubstrate as in the case of classical dehydrogenases. Several apparently unrelated enzymes such as
S-adenosylhomocysteinase (EC 3.3.1.1), dTDPglucose
oxidoreductase (EC 4.1.1.46), and UDP-glucuronate decarboxylase (EC
4.1.1.35) mechanistically belong to this new class of oxidoreductases.
In each case catalysis is initiated by reduction of enzyme-bound NAD.
The oxidized substrate then undergoes chemical transformation, if
necessary, on the enzyme surface before the hydride is
stereospecifically returned back and the product is released (1, 2).
In case of the epimerase, UDP-4-ketohexose and NADH have been
unambiguously demonstrated to be the reaction intermediates on the
enzyme surface. Stereospecific return of the hydride from NADH to the
opposite face of the oxidized hexose moiety completes the catalytic
process (1-7). Most of the studies on mechanism, quaternary structure,
and active site have been carried out with the Kluyveromyces
fragilis and Escherichia coli enzymes. Extensive chemical modification studies with the yeast enzyme revealed the requirement of one essential thiol (8, 9) one histidine (10), and an
arginine (11) in the overall catalytic process. The primary sequence
and the three-dimensional structure of this enzyme are not known. The
homodimeric 79-kDa E. coli holoenzyme, on the other hand,
has recently been cloned, expressed, and crystallized in various
catalytically inactive forms (12-14). Although no modification work
has been reported with the enzyme, a reasonably clear picture of the
active site has emerged at 1.8× resolution (13, 14). The substrate
analog UDP-benzene seems to be in a stretched conformation, and amino
acid residues involved in various binding interactions can be
tentatively identified. It is imperative that modification studies be
carried out with the E. coli enzyme to specify and confirm
the tentative roles of these amino acid residues residing at the active site.
In the previous paper we demonstrated that
uridine5'-diphosphoro- All biochemicals unless otherwise stated were purchased from
Sigma. [1-14C]Phenylglyoxal (2.17 mCi/mmol) was purchased
from Bhaba Atomic Research Center. Common chemicals were of analytical
grade and purchased from Merck.
Absorption measurements were done in a Hitachi U-3200
spectrophotometer, and fluorescence measurements were done in a Hitachi F-4010 spectrofluorimeter.
Enzymes and Assays--
Highly purified and essentially
homogenous UDP-glucose-4-epimerase was prepared from E. coli
according to the method of Wilson and Hogness (3). The specific
activity of the enzyme was 150-160 units/mg of protein, where 1 unit
of enzyme could convert 1 µmol of UDP-galactose to UDP-glucose in 1 min. Epimerase activity was routinely determined by the use of the
coupled assay system of Wilson and Hogness (3). In this case,
UDP-glucose, the product of epimerization, is immediately converted to
UDP-glucuronic acid by coupling the reaction with UDP-glucose
dehydrogenase and NAD. The assay mixture consisted of 0.1 M
glycylglycine buffer, pH 8.8, 0.25 mM NAD, 0.16 units of
UDP-glucose dehydrogenase, and the requisite amount of epimerase. The
reaction was initiated by addition of UDP-galactose, and increase in
absorbance attributable to formation of NADH was measured at 340 nm for
second and fifth minutes. Because the synthetic fluorophore itself had
a significant absorbance at 340 nm, a two-step assay system was used
whenever necessary. This assay has been described in detail earlier
(17). Briefly, in a total volume of 200 µl containing 0.2 M glycylglycine buffer, pH 8.8, and 30 µg of bovine serum
albumin, the requisite amount of epimerase was taken, and reaction was
initiated with UDP-galactose. The reaction was terminated after 5 min
by rapid addition of chloroform and vigorous shaking. The mixture was
then centrifuged. In the second stage, an appropriate aliquot from the
top aqueous layer was taken out to estimate the amount of UDP-glucose
formed by adding it in a medium containing UDP-glucose dehydrogenase
and NAD and observing the change in absorbance at 340 nm. A control
blank without epimerase was parallely run. Protein was estimated by the
method of Lowry et al. (18). For all stoichiometric calculations, the molecular weight for the epimerase was assumed to be
79,000 (12).
Synthetic Fluorophores and Fluorescence
Measurements--
UDPAmNS and other AmNS derivatives were synthesized
and purified according the methods described in our preceeding paper
(15). Wherever necessary, excess ligands and reagents were removed from reaction mixtures by Sephadex G-50 spin column centrifugation as
described by Maniatis et al. (19).
Modification Experiments--
Modification with phenyglyoxal was
carried out in 0.05 M potassium phosphate buffer, pH 8.0. The reagent was dissolved in dimethyl sulfoxide, which had no effect on
the stability of the enzyme. Modification with 1,2-cyclohexanedione and
2,3-butanedione was carried out in 0.05 M sodium borate
buffer, pH 8.8. Borate had no inhibitory effect on the enzyme. All
experiments, unless otherwise stated were carried out at 28 °C. All
kinetics of inactivation were followed by measurement of residual
activities of reaction mixtures, withdrawn at intervals and suitably
diluted. The modifying reagents at the concentrations used in the assay
medium did not have any effect on UDP-glucose dehydrogenase, the
coupling enzyme of the coupled assay procedure. In reactivation
experiments the inactivated epimerase was treated with neutralized 0.5 M hydroxylamine, which on dilution during assay had no
effect on the activity of the control enzyme.
Incorporation of
[14C]Phenylglyoxal--
[1-14C]
Phenylglyoxal incorporation studies were performed to determine the
stoichiometry of the reaction of phenylglyoxal with arginine residues
of the epimerase. The enzyme (0.6 mg/ml) in 0.5 M potassium
phosphate buffer, pH 8.0, was incubated with 2.5 mM
[1-14C] phenylglyoxal (1.2 × 106
cpm/µmol) for 45 min. Excess reagent and ligand were removed by spin
column centrifugation, and the eluates were measured for enzyme
activity, protein concentration, and radioactivity.
Estimation of Quantum Enhancement Q--
This was done according
the procedure of Mas and Colman (20) using the following equation:
1/(F/F0 UDPAmNS Is a Probe for the Active Site of E. coli
Epimerase--
We first investigated whether UDPAmNS can be used as a
probe for the active site of the E. coli enzyme. Both the
K. fragilis and the E. coli enzymes are known to
be competitively inhibited by UMP and UDP (21, 22). UDPAmNS was also
found to be a strict competitive inhibitor for the epimerase (Fig.
1) and hence a probe for the active site
of the enzyme. Clearly, substitution of the hexose moiety by the bulky
AmNS did not present any major steric problem for specific interaction
of this ligand to the substrate-binding site of the enzyme. This is
consistent with the reaction mechanism that assumes free rotation of
the ketohexose moiety during catalysis (2). Furthermore, earlier
observations had shown that considerable modification of the hexose
moiety or its substitution by other moieties can be effected without
hampering the binding property of the substrate (23-25). The
Ki for UDPAmNS was calculated to be 0.20 mM, which compares very well with the Ki
obtained with other such similar aromatic analogs of UDP such as
p-bromoacetamidophenyluridyl pyrophosphate (0.21 mM) and p-nitrophenyluridyl pyrophosphate (0.21 mM) (24, 25).
Conformational Transition of UDPAmNS on the Enzyme
Surface--
Fig. 2A shows
the fluorescence spectra of UDPAmNS (9.3 µM) as the
concentration of epimerase was progressively increased in the cuvette.
Quite evidently, the stacked and quenched fluorophore assumes a
stretched conformation on interaction with the enzyme and hence is
relieved of its quenching (15). Fig. 2A, inset, shows that
extrapolation at infinite enzyme concentration when all the
fluorophores have interacted with the enzyme results in a 8-fold
increase in fluorescence, a value that agrees excellently with the
value obtained for the fully unstacked fluorophore in isopropanol and
dimethylsulfoxide or after phosphodiesterase bond cleavage
(previous paper, Fig. 1; Ref. 15).
Fig. 2B shows complete displacement of UDPAmNS from the
enzyme surface by UDP. This is additional evidence to show that the fluorophore is interacting with the enzyme exclusively at the substrate
binding site. The native E. coli holoenzyme displays a weak
intrinsic fluorescence ( Modification Studies with
A specific test for modification of essential arginine residue(s) is
the regeneration of activity in presence of hydroxylamine (29). The
enzyme modified by cyclohexanedione and inactivated to <10% of its
original activity could be completely reactivated when the inactivated
enzyme was diluted 20-fold in 0.5 M neutralized hydroxylamine in 50 mM potassium phosphate buffer, pH 7.0 (data not shown). In contrast, when the enzyme was inactivated with phenylglyoxal, it could not be significantly reactivated. Such irreversible modification is indicative of the formation of a stable
diphenylglyoxal derivative (32). To establish the stoichiometry of
phenylglyoxal to arginine also as the number of arginine residues that
are modified during the process of inactivation, both
spectrophotometric analysis and radiolabeling with
[1-14C]phenylglyoxal were carried out. For this purpose,
epimerase (0.8 mg/ml) was incubated with phenylglyoxal (2.5 mM) in 0.05 mM potassium phosphate buffer, pH
8.0, for 45 min at 28 °C. The inactivated enzyme (>95%) was freed
of excess reagent in a spin column and a differential spectrum was
obtained with the untreated enzyme as the control. The differential
spectrum with
The spectral signal at 250 nm for the formation of the diphenylglyoxal
derivative provided a handle to directly relate the extent of arginine
modification with loss of activity. Fig.
4 shows that although all the three
arginine residues are reactive, the loss of activity versus
number of arginine residues modified extrapolates to 1 at zero activity
of the enzyme. The excellent correspondence between this direct method
and the kinetic method leaves little doubt that at least 1 arginine
residue is intimately associated with the overall catalytic process.
The usefulness of such a combination of methods was first demonstrated
by Hayman and Coleman (31) for isocitrate dehydrogenase.
Location of the Essential Arginine Residue--
Protection
experiments with UMP or UDP convincingly demonstrated that the
essential arginine residue is indeed located in the substrate binding
region of the active site. Fig. 5 shows that the rate of inactivation of phenylglyoxal is progressively decreased as the concentration of UMP is increased. A plot of t1/2 of inactivation against the concentration
of 5'-UMP was linear (Fig. 5, inset). The intercept on the
abscissa of such a plot gives the dissociation constant
(Kd) of the enzyme-protecting agent complex (27).
The Kd of the epimerase-UMP complex was calculated
to be 1.9 mM, which is in excellent agreement with the
Kd for UMP (1.9 mM) obtained kinetically
(22). The experiment with UDP also gave strong protection. Based on this protection experiment, the essential arginine residue was placed
in the binding subsite for the nucleotide phosphates.
The Essential Arginine Residue Is Critical for Stretching and
Binding of the Substrate--
To ascertain the specific role of the
essential arginine residue located at the uridylphosphoryl binding
subsite, the enzyme inactivated with phenylglyoxal (>95%) was allowed
to interact with UDPAmNS. Fig. 6 shows
that the quenched fluorophore failed to show significant enhancement of
fluorescence with the modified enzyme. Clearly, in this case, the
stacked fluorophore failed to bind and undergo transition to a
stretched conformation on the enzyme surface. In contrast, interaction
with the native enzyme that served as the control resulted in
severalfold enhancement of fluorescence of UDPAmNS. Furthermore,
UDPAmNS could be completely displaced from the enzyme surface by UDP
(Fig. 6, trace e), confirming its specific interaction at
the substrate binding site of the enzyme. The essential arginine
residue is, therefore, critically involved not only in the binding but
also in the destacking or stretching of this substrate analog on the
enzyme surface. The conformational change that the ligand underwent on
the enzyme surface could be monitored quite conveniently only because
this transition was accompanied by significant enhancement of
fluorescence attributable to release of stacking and consequential
absence of collisional quenching.
In the previous paper we had shown that when a uridine nucleotide
is derivatized with a suitable aromatic fluorophore such as AmNS via a
phosphoramidate bond through the terminal phosphate, it takes a stacked
conformation in aqueous solution that is in rapid equilibrium with its
extended form (15). The dynamic collisional quenching of fluorescence
in the stacked form should make such strongly quenched fluorophores
eminently suitable as probes for protein or enzyme studies provided
such designed molecules satisfy the following conditions. First, the
derivatized probe must be true structural analogs of the desired ligand
so that specificity is retained, and second, the probe on interaction
with the target protein should take a destacked conformation so that
the resultant enhancement of fluorescence can be a direct monitor of
the conformational transition of the probe and hence of its interaction
with the protein. Our present work shows that UDPAmNS satisfies both
the conditions when E. coli epimerase is used as the enzyme
target. Although the competitive nature of inhibition (Fig. 1) suggests it to be an active site-directed analog, maximum enhancement of fluorescence when all the molecules of the fluorophores are bound to
the enzyme (Fig. 2A) clearly demonstrates a destacked or
stretched conformation of the substrate analog on the enzyme surface.
These results agree very well with the published x-ray data of the
holoenzyme as it is co-crystallized with UDP-phenol or with other
abortive forms of the enzyme (12-14). Because the destacking energy
was calculated to be 2.3 Kcal/mol for UDPAmNS (previous paper; Ref. 15), the binding energy for the UDP moiety of the substrate may be
assumed to have a minimal value of that order.
Kinetic analysis with Fig. 7 provides a schematic
representation of the overall process. The stacked and quenched
fluorophore in the buffered aqueous solution assumes a stretched
conformation on the enzyme surface that leads to dequenching of
fluorescence. The essential arginine residue is obligatorily needed in
this binding process. These results fit exceedingly well with the
published x-ray and sequence homology data (13, 14). Resolution at
×2.0 of the holoenzyme co-crystallized with UDP had earlier shown that
apart from four molecules of water, Asn-179, Leu-200, Ala-216, and
Arg-292 are in a bonding distance of UDP. More importantly, the
oxyanion of -1-(5-sulfonic acid) naphthylamidate (UDPAmNS) is a
stacked and quenched fluorophore that shows severalfold enhancement of
fluorescence in a stretched conformation. UDPAmNS was found to be a
powerful competitive inhibitor (Ki = 0.2 mM) for UDP-glucose-4-epimerase from Escherichia
coli. This active site-directed fluorophore assumed a stretched
conformation on the enzyme surface, as was evidenced by full
enhancement of fluorescence in saturating enzyme concentration. Complete displacement of the fluorophore by UDP suggested it to bind to
the substrate binding site of the active site. Analysis of inactivation
kinetics in presence of
,
-diones such as phenylglyoxal, cyclohaxanedione, and 2,3-butadione suggested involvement of the essential arginine residue in the overall catalytic process. From spectral analysis, loss of activity could also be directly correlated with modification of only one arginine residue. Protection experiments with UDP showed the arginine residue to be located in the uridyl phosphate binding subsite. Unlike the native enzyme, the modified enzyme failed to show any enhancement of fluorescence with UDPAmNS clearly demonstrating the role of the essential arginine residue in
stretching and binding of the substrate. The potential usefulness of
such stacked and quenched nucleotide fluorophores has been discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-1-(5-sulfonic acid) naphthylamidate
(UDPAmNS)1 in aqueous
solution behaves as a quenched fluorophore because of its predominantly
stacked conformation. Quenching is fully released when complete
destacking takes place (15). To explore the possibility of whether such
designed fluorophores can be used to probe conformational transitions
of a putative ligand as it interacts with its target protein, we used
UDPAmNS as the probe and the E. coli epimerase as the model
target enzyme for this purpose. We first show that UDPAmNS is indeed a
substrate site-directed probe for this enzyme. Furthermore, the probe
assumes a fully stretched conformation on the enzyme surface, as is
evidenced by total dequenching of fluorescence on interaction with the
enzyme. Finally, we demonstrate the essential requirement of at least one arginine residue in this binding process. The reliability of these
results is confirmed from the available x-ray data.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1) = Kd/Q·n·P + 1/Q, where F0 and F
represent the fluorescence intensity of UDPAmNS in the absence and in
presence of the E. coli epimerase after subtracting that
attributable to epimerase, and P is the epimerase
concentration. The intercept of the plot of
1/(F/F0
1) versus
1/P gives 1/Q, where Q is the quantum
enhancement or the enhancement that would occur when all the
fluorophore is bound to the protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Inhibition kinetics of epimerase with
UDPAmNS. The inhibition kinetics was carried out by two-step assay
method as described under "Materials and Methods." UDPAmNS
concentrations for the different sets are no UDPAmNS ( ), 0.23 mM (
), and 0.46 mM (
). Blank was also
done containing no epimerase. The Ki for UDPAmNS was
0.20 mM.
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Fig. 2.
A, destacking of UDPAmNS on interaction
with epimerase. UDPAmNS (9.3 µM) and E. coli
epimerase (27.8 µM) were taken in 100 mM
potassium phosphate buffer, pH 8.0, and excited at 360 nm, and the
emission spectrum was scanned. It was then serially diluted with 9.3 µM UDPAmNS in the same buffer to vary the enzyme
concentration keeping the fluorophore concentration constant and
similarly scanned at every step. The spectra at different epimerase
concentrations (µM) in the presence of 9.3 µM UDPAmNS were: a, 27.83; b,
25.05; c, 22.26; d, 19.48; e, 16.70;
f, 13.91; g, 11.13; h, 8.35;
i, 5.57; and j, no epimerase. Inset,
plot of 1/(F/F0 1) against
1/P. Here F0 and F
represent the fluorescence intensity of UDPAmNS in the absence and
presence of the epimerase at an excitation wavelength of 360 nm after
subtracting that attributable to free protein. P is the
epimerase concentration. The intercept of the plot on the
1/(F/F0
1) axis gives
1/Q, where Q is the quantum enhancement or the
enhancement that would occur when all the fluorophore is bound to the
protein (see "Materials and Methods). B, displacement of
UDPAmNS from the epimerase by 5'-UDP. E. coli epimerase (20 µM) and UDPAmNS (9.3 µM) were taken in 200 µl of 100 mM potassium phosphate buffer, pH 8.0, and
excited at 360 nm, and the emission spectrum was taken (c).
To it were added small aliquots (1-10 µl) of concentrated stock
solution of 5'-UDP, and the spectrum was similarly scanned. The final
concentration of 5'-UDP are represented by 2 (d), 4 (e), 8 (f), and 16 (g)
µM and (h) 1 mM. The fluorescence spectra of
free enzyme and free UDPAmNS in buffer are represented by a
and b, respectively.
em = 453 nm) when excited at 360 nm. This is probably attributable to the varying amounts of NADH that
are known to be bound to the dimeric apoenzyme when purified from
overexpressed E. coli (26). We shall now show that by using this fluorophore one can uncover very conveniently amino acid residues
that are essential for the binding of the substrate.
,
-Diones--
Because the
substrate for epimerase is negatively charged at the cellular pH, we
decided to explore the possible involvement of cationic arginine
residue(s) in the binding of the substrate. We had earlier shown the
presence of an essential arginine residue at the substrate binding site
of the K. fragilis enzyme (11). Incubation of epimerase with
phenylglyoxal led to a progressive loss of enzyme activity. The
inactivation followed a pseudo-first order kinetics. A plot of
logK versus log of reagent concentration (27)
resulted in a straight line with a slope close to unity (Fig.
3A), indicating that at least
1 mol of phenylglyoxal/mol of enzyme was required to produce
inactivation. Because phenylglyoxal has occasionally been shown to
react with residues other than arginine (28), two highly selective
arginine-modifying reagents, cyclohexanedione (29) and butanedione
(30), were also used to modify the enzyme. In both cases phosphate
buffer was replaced by borate to stabilize the adducts (28). With
cyclohexanedione also, epimerase was completely inactivated following a
pseudo-first order kinetics, and a reaction order of 1 was obtained
(Fig. 3B). Similar results were obtained with
2,3-butanedione (data not shown). Notwithstanding some limitations in
this kinetic method (31), because the kinetic order of inactivation was
close to 1 in all cases, the minimal number of arginine residue(s) that
were involved in the inactivation process could be taken to be 1.
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Fig. 3.
A, inactivation of epimerase by
phenylglyoxal. The enzyme (7.5 µg/ml) was incubated with varying
concentrations of phenylglyoxal in 0.05 M potassium
phosphate buffer, pH 8.0, at 28 °C. At the indicated intervals,
aliquots were withdrawn for measuring enzyme activity. The
phenylglyoxal concentrations in the various runs were 0.25 mM ( ), 0.5 mM (
), 1 mM
(
), 2 mM (
) and 5 mM (
).
Inset, plot of puedo-first order rate constant
(K) versus log of phenylglyoxal concentration.
The slope of the plot is 0.75. B, inactivation of epimerase
by 1,2-cyclohexanedione. The enzyme (6 µg/ml) was incubated with
increasing concentrations of cyclohexanedione in 0.05 M
sodium borate buffer, pH 8.8. Aliquots were withdrawn at the indicated
intervals for measuring residual activity of the enzyme. The
cyclohexanedione concentrations at various runs were 1.25 mM (
), 2.5 mM (
), 5 mM (
), and 10 mM (
). Inset,
plot of log psuedo-first order rate constant (K) of
cyclohexanedione inactivation reaction versus log of
cyclohexanedione concentration. The slope of the plot is 0.8.
max at 250 nm was characteristic of a
diphenylglyoxal derivative (data not shown). Using a molar absorption
coefficient of 11,000 M/cm for the diderivative (32), 3.1 arginine residues were found to be modified in the process. In a
parallel experiment with [1-14C]phenylglyoxal, 5.8 mol of
phenylglyoxal was found to be incorporated per mole of the enzyme under
identical conditions, confirming the stoichiometry of phenylglyoxal to
arginine to be 2:1.
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Fig. 4.
Correlation between loss of activity and
number of arginine residues modified on phenylglyoxal treatment of the
epimerase. The enzyme (0.58 mg/ml) was treated with 2.5 mM phenylglyoxal in 0.05 M potassium phosphate
buffer, pH 8.0, containing 1 mM EDTA, at 28 °C for 60 min. At intervals of 5, 10, 15, 20, 30, 40, 50, and 60 min aliquots
were withdrawn and subjected to spin column centrifugation to terminate
the inactivation reaction. Small aliquots of the column eluates were
removed for activity measurement and protein estimation. The bulk of
the eluates were subjected to difference scan (230-330 nm) against
control enzyme treated in an identical fashion. Fractions of arginine
residues modified were determined from the molar extinction coefficient
for diphenylglyoxalated arginine at 250 nm ( = 11,000 M/cm).
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Fig. 5.
Protection against inactivation by
phenylglyoxal by uridyl phosphates. Epimerase (10 µg/ml) in 0.05 mM potassium phosphate buffer, pH 8.0, was treated with 2.5 mM phenylglyoxal in the absence or presence of increasing
concentrations of 5'-UMP. a-e, UMP (mM) concentrations of
0, 1.5, 3, 6, and 9, respectively; f, kinetics in the
presence of UDP (4 mM); g, untreated epimerase
control. Inset, plot of half-time of inactivation
(t1/2) against concentration of UMP. The slope
of the plot gives the Kd of the enzyme-UMP
complex.
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Fig. 6.
Interaction of UDPAmNS with native and
modified epimerase. E. coli epimerase (27 µM) was incubated with 5 mM phenylglyoxal in
100 mM potassium buffer, pH 8.0, for 2 h till complete
inactivation. It was then passed through a Sephadex G-50 spin column to
remove excess reagent. 27 µM unmodified enzyme was also
similarly passed through the spin column. At an excitation wavelength
of 360 nm the following spectra were taken in the same buffer as above:
a, unmodified epimerase (27 µM); b,
UDPAmNS (9.3 µM); c, unmodified epimerase (27 µM) and UDPAmNS (9.3 µM); d,
displacement of UDPAmNS with 10 µM UDP; e,
after total displacement of UDPAmNS with 1 mM UDP;
f, phenylglyoxal-treated epimerase (27 µM)
and UDPAmNS (9.3 µM).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
-dicarbonyl reagents (Fig. 3), followed
by estimation of arginine residues that can be directly correlated with
total loss of activity (Fig. 4), clearly shows that one arginine
residue is essential for the overall catalytic activity. Complete
regeneration of activity of 1,2-cyclohexanedione-inactivated enzyme by
hydroxylamine and of 2,3-butanedione-inactivated enzyme on removal of
borate by dilution (see text) is also consistent with modification of
an essential arginine residue on the enzyme surface. Nearly complete
protections provided both by UMP and by UDP against modification by
phenylglyoxal (Fig. 5) strongly suggest that the essential arginine
residue is located in the uridylphosphoryl binding subsite of the
active site and is probably involved in the productive binding of the
substrate with the enzyme. Using UDPAmNS as the active site-directed
designed probe, we could at this stage convincingly demonstrate that
the essential arginine residue is critically needed both for binding
and for stretching of the substrate (Fig. 6).
-phosphate of UDP has a potential bonding interaction
with the guanidino nitrogen of Arg-292. This, most likely, is the
essential arginine residue uncovered by our modification studies.
Interestingly, this arginine residue is conserved across the
phylogenetic scale for epimerase, which includes clones from several
organisms such as bacteria, yeast, and mammals (14).
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Fig. 7.
Schematic diagram depicting the binding of
UDPAmNS at the substrate binding site of E. coli
epimerase.
Our present study with UDPAmNS as a representative compound for stacked
and quenched fluorophores shows that such designed nucleotide
fluorophores can possibly be of considerable use in following
ligand-protein interactions in several classes of proteins of great
biological interest. Several ATPases, kinases, and G-proteins in
general show that nucleotides take a stretched conformation on the
protein surface (16, 33). In principle, the conformational transition
of such a quenched fluorophore as it interacts with its target protein
can thus be used to study many aspects of the structure-function
relationship of these enzymes or proteins. An anticipated advantage in
terms of ligand specificity for such synthetic fluorophores may be
attributable to the fact that both the base and the ribose moieties are
left unaltered during the derivatization process. Like other extrinsic
fluorophores, such fluorophores can also be used for energy transfer
studies, because the tryptophan emission spectrum overlaps the
absorption spectrum of these potential fluorophores. These fluorophores
can also be uniquely useful as sensors for the formation of substrate
binding or ligand binding sites in protein-folding studies. Finally,
search for lead compounds for drug development may be facilitated by rapid fluorimetric monitoring of the displacement of stretched fluorophores from the binding site of target proteins, e.g.
G-proteins, by an array of synthetic molecules generated by
combinatorial methods or extracts from plant sources.
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FOOTNOTES |
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* This work was supported in part by the Council for Scientific and Industrial Research (CSIR), India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Molecular Pharmacology, Albert Einstein
College of Medicine, New York, NY.
§ Research Associate for the CSIR. Present address: Dept. of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095.
¶ Emeritus Scientist for the CSIR. To whom correspondence should be addressed: Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Rd., Jadavpur, Calcutta, India 700032. Tel. and Fax: 91-33-4735197; E-mail: IICHBIO{at}GIASCL01.VSNL.NET.IN.
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ABBREVIATIONS |
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The abbreviation used is:
UDPAmNS, uridine-5'-diphosphoro--1-(5-sulfonic acid) naphthylamidate.
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