(Received for publication, November 2, 1994; and in revised form, April 24, 1995)
From the
S-Adenosylmethionine (AdoMet) synthetase catalyzes the
formation of AdoMet from ATP and L-methionine with subsequent
hydrolysis of the bound tripolyphosphate intermediate. Maximal activity
requires the presence of two divalent and one monovalent cation per
active site. Recently, the x-ray structure of the Escherichia coli AdoMet synthetase was solved, and the positions of the two
Mg
S-Adenosylmethionine synthetase
(ATP:L-methionine S-adenosyltransferase or AdoMet (
Figure SI:
Scheme IThe two sequential reactions
catalyzed by AdoMet synthetase.
The crystal structure
of E. coli metK-encoded AdoMet synthetase isozyme has been
solved at 3-Å resolution by Takusagawa and co-workers. ( We report
here results that confirm an important role of glutamate 42 in
monovalent cation activation. These studies, in combination with the
crystallographic data, suggest that similar structural modes of
potassium activation may exist between AdoMet synthetase and two other
K
The stability constants of
UO
The mutant with
lysine at position 42, E42KMetK, was also constructed in order to
determine whether the change in side-chain charge might result in an
enzyme that was fully active in the absence of a monovalent cation.
However, the E42KMetK mutant protein did not accumulate in
vivo, suggesting misfolding and proteolytic degradation. Thus the
E42KMetK mutant was not amenable to characterization.
Figure 1:
KCl activation of the rate of AdoMet
formation by wild type MetK and E42QMetK. Reactions were carried out at
22 °C and contained Tris
We first
examined whether the UO Many tight-binding
inhibitors are also slow-binding inhibitors (19) and require
specialized kinetic analysis. Slow onset of inhibition is only relevant
to the kinetic analysis if it is slow enough that the reaction time
courses do not reflect the true steady state rates. The possibility of
slow binding was evaluated by following the product formation from
partially inhibited enzyme for 10 min, followed by addition of an
additional 20 µM UO
Figure 2:
Time course of the onset of uranyl
inhibition. Solutions contained 100 mM
ACES
Plots of 1/Vversus 1/KCl at various
concentrations of UO
Figure 3:
Double-reciprocal plots of uranyl
inhibition versus KCl (a) and ATP (b).
Solutions contained 100 mM
ACES
The plot of 1/Vversus 1/ATP at various UO
Figure 4:
Velocity of AdoMet formation as a function
of AdoMet synthetase concentration in the presence of uranyl acetate.
Solutions contained 100 mM
ACES
In (and , see below), R represents the reciprocal of the steady
state velocity in the absence of inhibitor, S is the
apparent inhibition constant, I is the inhibitor
concentration, E is the concentration of total protein,
and When the data were fit with , the best fit
resulted in a value of 10 for The nonlinear least squares fit from an experiment in which
enzyme was varied at constant uranyl concentration of the data to is shown in Fig. 4. The fit gave a value for the
apparent inhibition constant of 510 nM (± 60
nM) with an R
Figure 5:
Fluorescence emission spectra of uranyl
cation, free and in the presence of ATP or tripolyphosphate. Solutions
contained 1 mM UO
The stability constants of
UO
The construction of the E42QMetK mutant was motivated by the
observation that the UO Uranyl was found to be a potent inhibitor of AdoMet
synthetase and a competitive inhibitor with respect to
K Although glutamate 42 is
necessary for K Why uranyl, a divalent
cation, binds tightly at the K
Finally, it is useful to make some comparisons between the
K
binding sites were identified. Based on additional
spherical electron density, the K
binding site was
postulated to be a nearby site where the uranyl heavy atom derivative
also bound in the crystal. The side chain of glutamate 42 is within
ligation distance of the metals. Mutagenesis of glutamate 42 to
glutamine (E42QMetK) abolished monovalent cation activation and
produced an enzyme that has kinetic properties virtually identical to
those of K
-free wild type AdoMet synthetase in both
the overall AdoMet synthetase reaction and in the hydrolysis of
tripolyphosphate. Thus, there is a
100-fold decrease in the V
for AdoMet synthesis and large increases in
the K
values for both substrates. In
contrast there is only a 2-fold decrease in V
for tripolyphosphate hydrolysis. The uranyl ion,
UO
, is a competitive inhibitor with
respect to K
(K
=
350 nM) and is the first ion to bind at this site and inhibit
the enzyme. The UO
inhibition is
reversible and tight-binding, and results from
UO
and not
UO
ATP. Analogous to K
activation, UO
predominantly
inhibits AdoMet formation rather than tripolyphosphate hydrolysis. The
kinetic results indicate that UO
inhibition is likely to result from interference with productive
ATP binding. UO
remains a tight-binding
inhibitor of the E42Q mutant, which suggests that K
and UO
have different ligation
preferences when bound in the monovalent cation binding pocket. The
results support the model that glutamate 42 provides ligands to the
K
and has a major role in monovalent cation binding.
)synthetase) is one of many enzymes that are activated by
monovalent metal cations(1) . However, the modes and structural
basis for monovalent cation activation are not well understood for most
of these enzymes. We have studied monovalent cation activation of
AdoMet synthetase in an attempt to define the structural mechanisms
involved in activation. S-Adenosylmethionine synthetase
catalyzes the two sequential reactions shown in Fig. SI(2) . The first reaction, AdoMet formation and
the second reaction, tripolyphosphate (PPP
) hydrolysis,
occur at the same active site. Both steps require divalent metal ions
such as Mg
. Two divalent metal ions bind per subunit
(active site) of the tetrameric enzyme from Escherichia coli and both are required for activity(3) . One monovalent
cation, K
or one of similar size, binds per active
site and stimulates the AdoMet formation step up to 100-fold at low
substrate concentrations, decreases the K
of ATP (4-fold) and L-methionine (12-fold), but has
relatively little effect (2-5-fold) on the rate of the PPP
hydrolysis reaction. The size of the monovalent cation dictates
how well it binds to, and activates, AdoMet synthetase. Spectroscopic
studies have shown all three metals bind close together in the active
site(4) .
Tl
NMR studies of the
enzyme Tl
complex suggest that the monovalent metal
activator does not coordinate directly to either substrates or
products(5) . Kinetic isotope effects indicate that the
monovalent cation does not alter the transition state structure, but
does enhance the commitment to catalysis of bound ATP(6) .
Based upon these results, monovalent cation activation has been
proposed to result from subtle alterations in protein
conformation(5) . The nature of these changes remains elusive,
but details about the structure of the K
binding site
and how it could affect the protein or active site conformation are now
starting to emerge, primarily as a result of the recently determined
x-ray crystal structure of AdoMet synthetase.
)The location of the two divalent metal ion binding sites
have been identified from differences in electron density maps of
enzyme crystals grown in the presence of either Mg
or
Co
. A third distinct metal ion binding site, 5 and 7
Å away from the two Mg
sites, was noted as a
site of other spherical high electron density. This was also the site
where UO
bound and was postulated to be
the K
binding site. This site contains the side chain
of glutamate 42 as a potential ligand to the metal ion. The only other
residue close to the K
(or uranyl) ion was serine 263.
These residues are conserved in all 11 reported AdoMet synthetase
sequences. Guided by the x-ray data, two sets of experiments were
conducted to test the importance of glutamate 42 as a ligand to the
activating K
ion: 1) site-directed mutagenesis of this
residue was performed to assess whether there was disruption of
K
activation, and 2) evaluation of whether
UO
activates like K
or
inhibits competitively with respect to K
.
-activated enzymes for which crystal structures have
recently been published, dialkylglycine decarboxylase (7) and
pyruvate kinase(8) .
Materials
AdoMet synthetase (wild type) was
purified by published methods(2) . Uranium oxyacetate (uranyl
acetate) dihydrate was purchased from NOAH and was of the highest
available purity. L-[methyl-C]methionine
(40-60 mCi/mmol, 16.9 mM) was purchased from DuPont NEN.
ACES, Tris, EDTA, ATP, Na
P
O
,
8-hydroxyquinoline, lysozyme,
isopropyl-
-D-thiogalactopyranoside,
-mercaptoethanol, streptomycin sulfate, and S-adenosyl-L-methionine (chloride form), were
purchased from Sigma. HEPES and MES were purchased from U. S.
Biochemical Corp. All other reagents were the highest purity available.
P-81 ion exchange paper used in enzyme assays was obtained from
Whatman.
Mutagenesis
Site-directed mutagenesis was done
using the Muta-Gene in vitro mutagenesis kit (Bio-Rad),
employing T4 DNA ligase, T4 DNA polymerase, and polynucleotide kinase
from New England Biolabs. Mutagenesis was performed on the plasmid
pT7-6(metK), which consists of the metK gene (9) inserted between the PstI and EcoRI sites
of pT7-6. ()The oligonucleotide (antisense strand) used in
mutagenesis was as follows: 5`-GCG CAA CGA ACG *GTT TGG ATG CAT TTT
TC-3`, where the asterisk is to the left of the mutated base. Plasmids
were purified by either Rapid Pure Miniprep (RPM-60, Bio101 Inc.) or
QIAGEN Plasmid Prep. (QIAGEN, Inc.). Following mutagenesis the plasmid
was transformed into E. coli strain DM22 (9) for
propagation. The DNA sequence of the mutated gene was verified by DNA
sequencing using the Sequenase kit (U. S. Biochemical Corp.). The
mutagenized plasmid pT7-6(metKE42Q) was ultimately transformed
by electroporation into strain RSR15(DE3), an E. coli B strain
for which extracts have no detectable AdoMet synthetase activity due to
a chromosomal mutation.
A mutant with a lysine at position
42 was prepared in an analogous fashion, yielding
pT7-6(metKE42K).
Purification of E42QMetK
E. coli strain
RSR15(DE3) containing the mutagenized plasmid was grown in LB medium
containing ampicillin (100 µg/ml) to an absorbance at 550 nm of
1.0, at which time 1 mM
isopropyl--D-thiogalactopyranoside (IPTG) was added.
RSR15(DE3)pT7-6(metKE42Q) cells were harvested after 4 h and
frozen at -80 °C. Cells were suspended in 0.1 M Tris
HCl, 1 mM EDTA, 0.1%
-mercaptoethanol, pH
8, and lysed by addition of lysozyme (1.0 mg/g cells) followed by
sonication. The protein purification followed previously described
methods(2) . The cell free homogenate was treated with
streptomycin sulfate, followed by ammonium sulfate fractionation.
E42QMetK-AdoMet synthetase was purified by chromatography on
phenyl-Sepharose (Sigma), hydroxylapatite (Bio-Rad), and
aminohexyl-Sepharose-4B (Pharmacia). Protein purity and native
structure were determined by denaturing and non-denaturing gel
electrophoresis. The protein concentration was determined by absorbance
at 280 nm (an A
of 1.3 represents a 1.0 mg/ml
solution of the enzyme). In analogous trials, the E42KMetK mutant did
not appreciably accumulate after IPTG induction, apparently as a result
of proteolysis. Thus E42KMetK-AdoMet synthetase was not further
characterized.
AdoMet Synthesis Assay
The overall AdoMet
synthetase activity was determined by methods described previously (2) with some modifications. The reaction mixture (50 µl)
contained 50 mM HEPESN(CH
)
OH, pH
8.0, 20 mM MgCl
, 0.1 mM ATP, L-[methyl-
C]methionine
(30-60 mCi/mmol, 0.1 mM), and where indicated 50 mM KCl. The reactions were terminated by the addition of 150 µl
of 25 mM EDTA, and 150 µl of the reaction was spotted on a
two-centimeter disk of P-81 filter paper (Whatman). After air drying, a
group of disks were placed on a Buchner funnel and washed with 4 liters
of distilled water; the retained radioactivity was quantified by liquid
scintillation counting in EcoScint scintillation mixture using a
Beckman LS2800 liquid scintillation counter. The effects of uranyl ion
on AdoMet formation activity were determined under identical
conditions, with the exception that the buffer was changed from
HEPES
N(CH
)
OH, pH 8.0, to 100 mM ACES
N(CH
)
OH, pH 6.2, and the
reactions were terminated by the addition of 150 µl of 25 mM EDTA, 100 mM Tris
HCl, pH 8.0. Substrate saturation
data were computer-fitted using the programs of Cleland(10) .
Other data were analyzed by least squares fitting in a variety of
computer programs.
Tripolyphosphate Hydrolysis Assay
The
tripolyphosphate hydrolysis activity was determined as described
previously(2) . Reactions (50 µl) consisted of 50
mM HEPESN(CH
)
OH, pH 8.0, 20
mM MgCl
, 0.1 mM Na
P
O
, and, where indicated,
0.1 mM AdoMet and 10 mM KCl. Phosphate formed as
product was quantified by a molybdate-malachite green
assay(11) . Tripolyphosphatase activity in the uranyl
inhibition studies was determined by the same method with the exception
that the buffer was changed from
HEPES
N(CH
)
OH to 100 mM ACES
N(CH
)
OH, pH 6.2, and the
reactions were terminated by the addition of 150 µl of 25 mM EDTA, 100 mM Tris
HCl, pH 8.0.
Stability Constants
Stability constants for
UOATP,
UO
PPP
,
Mg
ATP, and Mg
PPP
at pH 6.2 were measured by electron paramagnetic resonance (EPR)
techniques (12, 13) using a Varian E-109 spectrometer.
The stability constants for Mn
ATP and
Mn
PPP
at pH 6.2 were determined by
the addition of ATP or PPP
to a solution of 0.1 mM MnCl
in 100 mM ACES
N(CH
)
OH, pH 6.2, which resulted
in a decrease in the EPR signal of free
Mn
(13) . The dissociation constant was
determined from the concentration of ATP or PPP
that
decreased the signal to half its total intensity, giving K
values of 92 and 57 µM,
respectively. The Mn
ATP and
Mn
PPP
complexes (0.1 mM MnCl
, and 0.1 mM PPP
or 0.4
mM ATP) were then titrated with a solution of
UO
(Ac)
, which led to the release of
Mn
and an increase in the EPR signal. Values for K
for uranyl complexes
were determined from the concentration of uranyl acetate, which led to
recovery of half of the total EPR signal; the actual K
value was calculated from the
relationship, K
= K
/(1 + A/K
), where A is the
concentration of the competing metal (Mn
), and K
is the dissociation constant for the
(Mn
ATP or
Mn
PPP
) complex (12) .
Analogous experiments were performed to obtain the K
for Mg
ATP (0.30 mM) and
Mg
PPP
(47 µM) under
these conditions.
ATP and
UO
PPP
were also
determined colorimetrically using 8-hydroxyquinoline as a
metallochromic dye source(12) . A solution of 0.4 mM 8-hydroxyquinoline in ACES
N(CH
)
OH pH
6.2, was titrated with a solution of UO
(Ac)
(0-60 µM final concentrations), which resulted
in an increase in absorbance at 380 nm. This result was then compared
to identical titrations of mixtures of 8-hydroxyquinoline and either
0.1 mM ATP or 0.1 mM PPP
. The K
values were calculated using: K
=
[UO
]
[ligand]
/[UO
ligand],
where ligand is ATP or PPP
(12) . K
values of 0.2 mM for
UO
ATP and 0.07 mM
UO
PPP
were obtained.
Fluorescence Spectra of UO
Solutions contained
100 mM ACES Complexes with ATP or PPP
N(CH
)
, pH 6.2 and 1
mM UO
(Ac)
. The spectra for
UO
ATP and
UO
PPP
were obtained by
the addition of either 2 mM ATP or 2 mM PPP
. The solutions were excited by 422 nm light with a
5-nm excitation slit width; the fluorescence emission spectrum was
detected with a 5-nm slit width. A Perkin-Elmer LS-50 luminescence
spectrometer was used. The final spectra are the average of three
scans.
Mutagenesis of Glutamate 42
The crystal
structure of AdoMet synthetase implicated glutamate 42 as a ligand to
the K activator. Glutamate 42 is the only charged
ligand in the vicinity of the K
binding pocket. Thus
the conservative mutation of glutamate 42 to glutamine (E42QMetK) might
disrupt monovalent cation binding and activation by removing the
negative charge. The E42QMetK mutant was therefore prepared and
characterized. During column chromatography, E42QMetK behaved
indistinguishably from wild type AdoMet synthetase. Pure E42QMetK is
indistinguishable from wild type AdoMet synthetase by electrophoresis
under denaturing and non-denaturing conditions. However, the specific
activity of the mutant is only 1% that of the
K
-activated wild type enzyme.
Kinetic Analysis of E42QMetK
Initially
K activation of E42QMetK was tested. Monovalent cation
activation of wild type AdoMet synthetase is most pronounced at low
substrate concentrations (
K
values,
0.1 mM for ATP, 0.1 mM for L-methionine).
The effect of KCl concentration on the activity of wild type versus E42QMetK is shown in Fig. 1. Unlike wild type AdoMet
synthetase, the activity of E42QMetK is not stimulated by KCl. The
comparison of steady state kinetic parameters for wild type and
E42QMetK is shown in Table 1; the results indicate that E42QMetK
behaves nearly identically to K
-free wild type AdoMet
synthetase in terms of K
and V
values as well as relative activities.
Furthermore, the K
for Mg
is not detrimentally affected by the mutation, consistent with
the crystallographic data, which indicate that glutamate 42 is not part
of the Mg
binding site. The results suggest that
glutamate 42 is essential for monovalent cation activation of AdoMet
synthetase, presumably by ligating K
. The E42QMetK
mutant was also not activated by 0.1 M LiCl, NH
Cl,
NaCl, or CsCl, in contrast to the wild type enzyme. Unfortunately there
is no direct method for measuring K
binding to AdoMet
synthetase, so it is not clear whether the mutation affects binding as
well as activation.
HCl (100 mM), pH 8.0. The
ATP, L-methionine, and MgCl
concentrations were
0.1, 0.1, and 20 mM, respectively.
Uranyl Inhibition of AdoMet Synthetase
Since
UO binds to glutamate 42 in crystals, we
investigated the effect of UO
on wild
type AdoMet synthetase activity. If glutamate 42 is a ligand to
K
, then UO
would be
expected to either activate analogously to K
or
competitively inhibit. No ions had been found to be competitive
inhibitors with respect to K
, i.e. all
monovalent cations tested either activate or apparently do not
bind(2) .
Uranyl Is a Reversible Tight-binding Inhibitor of AdoMet
Synthetase
Experiments with uranyl acetate were carried out at
pH 6.2 since at higher pH polynuclear UO species and ``uranyl and amorphous hydroxide''
precipitates form(14, 15) . Uranyl acetate was soluble
up to
pH 6.5 in the normal AdoMet synthetase assay buffer,
therefore to diminish problems associated with the chemical instability
of UO
, experiments were performed at pH
6.2. The kinetic constants for AdoMet synthetase at pH 6.2 are
comparable to those at pH 8.0 (see below). The uranyl cation was found
to be a potent inhibitor of AdoMet synthetase. Uranyl acetate at 25
µM fully inhibits K
-free AdoMet
synthetase in the presence of K
concentrations of substrates. The inhibition was eliminated
by the addition of 10 mM KCl but was unaffected by the
addition of up to 100 mM MgCl
, consistent with
uranyl binding at the monovalent cation binding site. The relatively
small amount of UO
required to inhibit
the enzyme suggested tight-binding inhibition, which needed to be
further characterized before steady state kinetic methods could be used
to determine if UO
is a competitive
inhibitor with respect to K
(16) . Two
conditions must hold true for complete steady state kinetic analysis of
tight-binding inhibitors; the inhibition must be reversible, and the
onset of inhibition must be fast with respect to the assay
used(16) . Preferably, the inhibitor concentration used should
be at least 10-fold higher than the enzyme concentration to avoid
complications associated with depletion of free inhibitor.
inhibition was
reversible. Irreversible inhibition was a particular concern since
uranyl is photo-reactive (17, 36, 37, 38, 39) and
could produce radical species in the active site resulting in
irreversible modification(18, 40) . For AdoMet
synthetase the uranyl inhibition was fully reversed by dilution into a
solution containing saturating amounts of potassium. This was shown by
an experiment in which 10 µM enzyme was incubated for 30
min with 50 µM UO
(Ac)
and
substrates (0.1 mM each) in a potassium-free buffer and then
the activity determined; the enzyme had 20% of the activity of an
UO
-free control. Parallel
uranyl-containing reaction mixtures were incubated for 30 min and then
diluted 10-fold into buffer containing 50 mM KCl, and further
incubated for 30 min prior to determination of the enzymatic activity.
The enzyme from the potassium-containing reaction mixture had 92% of
the activity of a uranyl free uninhibited control, thus demonstrating
that uranyl inhibition was reversible.
(Fig. 2). There was no observable lag in the onset of
additional inhibition (Fig. 2)(19) . Since the uranyl
inhibition was both reversible and had a rapid onset, conventional
steady state kinetic methods were used to determine the inhibition
patterns with various substrates.
N(CH
)
OH pH 6.2, 20 mM MgCl
, 0.1 mM ATP, 0.1 mML-methionine, 1 µM AdoMet synthetase active
sites, and 10 µM UO
(Ac)
, for the
first 15 min. After 15 min, additional UO
(Ac)
was added to a final concentration of 30 µM. The
reaction was carried out at 22 °C. The lines are the best fits to
the data from 0 to 15 min and 15 to 25 min.
Characterization of Uranyl Inhibition by Steady State
Kinetics
Double reciprocal plots were used to reveal the type of
inhibition with respect to K, ATP, and methionine.
Initially, kinetic constants for AdoMet synthetase at pH 6.2 (100
mM ACES
N(CH
)
OH, 20 mM MgCl
, and 50 mM KCl) were determined to
examine whether the enzyme activity was altered at this lower pH.
Compared to the standard assay conditions at pH 8.0, the K
values for ATP (0.11 ± 0.01) and L-methionine (0.10 ± 0.01 mM) are essentially
unchanged, and the V
is reduced only 2-fold.
intersect on the
1/V axis and thus show that uranyl is a competitive inhibitor
with respect to potassium (Fig. 3a). A true inhibition
constant could not be extracted from a replot of slope versus inhibitor concentration because the replot is not linear (Fig. 3a, inset), which was expected since the
concentration of inhibitory species is comparable to the enzyme
concentration (see below). An alternative method presented below was
used to determine the inhibition constant. Nevertheless, these results
demonstrate that UO
is a competitive
inhibitor with respect to potassium, therefore confirming that both
UO
and K
bind at the
same site. The double-reciprocal plot of initial velocity versusL-methionine concentration at four different levels of
UO
showed a noncompetitive pattern with
intersection on the abscissa (data not shown).
N(CH
)
OH, pH 6.2, 20 mM MgCl
, and, for a, 0.1 mM ATP, 0.1 mML-methionine, 1 µM AdoMet synthetase active sites, and 0, 4, 6, 8, and 10 µM UO
(Ac)
. The reactions for b contained 100 mM ACES
N(CH
)
OH, pH 6.2, 20 mM MgCl
, 0.1 mML-methionine, 1
µM AdoMet synthetase active sites and 0, 10, 15, and 20
µM UO
(Ac)
. All reactions were
carried out at 22 °C.
concentrations should reveal whether free
UO
or
UO
ATP is the predominant inhibitory
species. Lanthanide metal ions, which inhibit of some
nucleotide-utilizing enzymes (e.g. yeast phosphoglycerate
kinase (20) and creatine kinase(16) ) are not
inhibitory as the free ion but are inhibitory as a
Ln
nucleotide complex. It is also known that
UO
binds to ATP(24) . If
UO
ATP is the inhibitory species one
would expect to see a set of downward curved lines intersecting at the
1/V axis, as was observed in studies of
Eu
ATP inhibition of yeast phosphoglycerate
kinase (20) and creatine kinase(16) . The downward
curvature results from increased concentrations of the inhibitory
Eu
ATP species as the total concentrations of
both ATP and Eu
increase. For AdoMet synthetase the
plot of 1/Vversus 1/ATP at fixed concentrations of
UO
shows the opposite trend (Fig. 3b), with a set of upward curved lines
intersecting at the 1/V axis, demonstrating competitive
inhibition. Reasoning analogous to the case of
Eu
ATP suggests that
UO
ATP is not the inhibitor, since
at higher concentrations of both ATP and UO
less inhibition is observed than expected from the low
concentration data. Rather, it is likely that ATP competes with the
enzyme for UO
.
Characterization of Tight-binding
Inhibition
Inhibition constants for tight-binding inhibitors are
typically obtained by methods outlined by Williams and
Morrison(16) . The methods involve measuring initial velocity
as a function of enzyme active site concentration at fixed
concentrations of inhibitor and substrates. If the inhibitor is a
tight-binding inhibitor such plots have upward curved lines, whereas
for conventional inhibitors the plots are linear. For AdoMet synthetase
the initial velocity was measured as a function of uranyl
concentration, and a non-linear relationship was found (Fig. 4).
When the data are fit to of Williams and Morrison, values
for K and the purity of
the enzyme can be
obtained(16) .
N(CH
)
OH, pH 6.2, 20 mM MgCl
, 0.1 mM ATP, 0.1 mML-methionine, in the presence or absence of 20 µM UO
(Ac)
(by mass, 3.5 µM inhibitory species as determined using ). The line with the
is the best fit to the data in the presence of
uranyl acetate using , K
= 510
nM. The
reflect data obtained in the absence of uranyl
acetate. Reactions were carried out at 22
°C.
is the fraction of total protein that can react with
inhibitor.
, suggesting there was 10
times more active enzyme in the reaction than had been introduced.
However, an alternative interpretation is a decrease in inhibitor
concentration, which is reasonable for UO
due to the multiple forms present in
solution(16, 18, 40) . can be
rewritten so that
reflects the fraction of total inhibitor
that can react with a known quantity of enzyme, yielding .
value of 0.99. The true
inhibition constant of 350 nM can be calculated using , the observation that UO
binds to ATP, and the measured stability constant for
UO
ATP of 0.22 mM (discussed below). The calculated concentration of inhibitory
uranyl,
I, was 3.5 µM (18 ± 4%
of the total). The difference in total (20 µM) versus inhibitory (3.5 µM) UO
may be attributed to the multitude of uranyl complexes present, e.g. the slowly interconverting polymeric hydrated uranyl
species, [(UO
)
(OH)]
and
[(UO
)
(OH)
]
,
and chelation of uranyl to ATP. As noted earlier,
UO
is stable only at low pH, and
polynuclear UO
species as well as
``UO
hydroxide'' form and
precipitate at the higher pH(14, 15, 40) .
O NMR and Raman spectroscopic studies have demonstrated
the existence of the polymeric hydrated uranyl species
[(UO
)
(OH)]
and
[(UO
)
(OH)
]
and that they can represent the majority of uranyl in aqueous
solution at pH values greater than 3.5(21, 22) . Based
upon the observation that a single uranyl ion binds to glutamate 42 in
the active site of the crystalline enzyme, we deduce that the free
UO
, and not one of the polymeric species,
is the actual inhibitor. Due to the large number of interconverting
complexes, it was not feasible to decipher the complete distribution of
the uranyl species.
Characterization of UO
To determine the affinity of ATP for
uranyl under our reaction conditions, we carried out fluorescence
studies. Uranyl is known to be chelated by polyphosphates(24) .
The fluorescence emission spectrum of the uranyl cation is extremely
sensitive to the type of
ligation(17, 23, 25, 36, 37, 38, 39) .
Titration of 1 mM UO2+-ATP and
UO
2+-PPP
Interactions
with
PPP
revealed formation of a 1:1 complex having a three-peak
emission spectrum (Fig. 5). In contrast, titration of
UO
with ATP produced a complex of
UO
ATP that has a single emission
peak at 490 nm that is clearly different than the emission spectrum of
the UO
PPP
complex. The
difference in the fluorescence emission patterns of
UO
ATP and
UO
PPP
suggest that the
structures of the complexes are different. Feldman et al. reported that the UO
ATP
complex involves chelation of UO
by the
N
atom of the adenine ring, the ribose 04` oxygen, and the
negatively charged oxygen of the
-phosphoryl group(24) .
Based upon their proposal that the linear triatomic structure of
UO
prevents tridentate ligation by the
polyphosphate chain (24) and the fact that the
UO
PP
fluorescence
emission spectrum is very similar to that of
UO
PPP
(data not shown),
it appears that the 1:1 UO
PPP
complex involves bidentate ligation. These results indicate that
UO
complexes with ATP and PPP
do form and have stability constants which are less than 1
mM.
(Ac)
, 100 mM
ACES
N(CH
)
, pH 6.2, and where present, 2.0
mM ATP or 2.0 mM
PPP
.
ATP and
UO
PPP
were determined
by EPR and colorimetric techniques (12, 13) (see
``Experimental Procedures''). The EPR technique, using
Mn
as a probe, involved titration of
Mn
-chelated ATP (K
= 0.092
mM) and Mn
-chelated PPP
(K
= 0.057
mM) complexes with UO
. Uranyl
binding to ATP or PPP
caused the release of Mn
from the ATP or PPP
complexes and an increase in the
EPR signal due to free Mn
. The apparent K
were 0.22 mM for
UO
ATP and 0.047 mM for
UO
PPP
. The stability
constants of UO
ATP and
UO
PPP
were also
determined by UV spectroscopic titration of a
UO
8-hydroxyquinoline (12) complex with the polyphosphates; these experiments gave
stability constants of 0.20 mM for
UO
ATP and 0.07 mM for
UO
PPP
, which agree well
with the values obtained by EPR. The stability constant of
UO
ATP confirms that ATP does
chelate UO
under the conditions of our
kinetic studies and thus ATP decreases the concentration of free uranyl
species in solution.
UO
The monovalent
cation activator exerts its stimulatory effect predominantly on the
AdoMet formation step and not the subsequent PPP2+ Inhibits AdoMet Formation and
Not PPP
Hydrolysis
hydrolysis
step. If UO
is a monovalent cation
antagonist, it might inhibit only the AdoMet formation step and not
PPP
hydrolysis. Consistent with the role of
UO
as a K
antagonist,
concentrations of uranyl that completely inhibit the overall reaction
had little effect on the rate of PPP
hydrolysis (Table 2). The lack of inhibition of the tripolyphosphatase
reaction cannot be attributed to a UO
sequestering effect of PPP
based upon the stability
constants of UO
PPP
and
Mg
PPP
complexes.
UO
PPP
and
Mg
PPP
have indistinguishable
dissociation constants of 47 µM (as determined by EPR
techniques). Therefore, under the assay conditions, 20 mM MgCl
, and 0.050 mM uranyl acetate, very
little of the uranyl will be sequestered in the form of
UO
PPP
. From these
results we conclude that uranyl inhibition, analogous to K
activation, exerts its effect predominantly on the first
half-reaction.
UO
Guided by the evidence that
UOInhibits Binding of
the Adenosyl Moiety
binds tightly and reversibly at the
potassium site, uranyl inhibition was used to probe the role of
potassium in activation of AdoMet formation. The effect of
UO
on nucleotide binding to AdoMet
synthetase was assessed by determining whether the hydrolysis of the
ATP analog A(S)TP is inhibited by UO
.
A(S)TP is an ATP analog in which the linkage between C5` and the
phosphoryl group contains sulfur rather than
oxygen(2, 26) . A(S)TP binds to AdoMet synthetase and
is hydrolyzed to yield A(S)DP and P
at a rate comparable to
the maximal rate of tripolyphosphate hydrolysis, 1000-fold faster than
ATP hydrolysis(2) . However, A(S)TP does not react with L-methionine to form AdoMet. Apparently when A(S)TP is bound
to AdoMet synthetase, it is positioned in such a way that the
-phosphoryl group is poised for hydrolysis, perhaps due to the
longer C-S-P bonds. If UO
only inhibits
the AdoMet-forming step, then A(S)TP may still bind and be hydrolyzed
by UO
-inhibited AdoMet synthetase,
whereas if UO
prevents productive ATP
binding, then A(S)TP would not be hydrolyzed by
UO
-inhibited AdoMet synthetase. The
results summarized in Table 2indicate that A(S)TP hydrolysis is
significantly inhibited by UO
, which
suggests UO
inhibits productive binding
of A(S)TP and ATP. Since increasing concentrations of A(S)TP will
overcome the inhibition by removing uranyl from the enzyme, as does
ATP, it is not possible to determine whether uranyl prevents A(S)TP
binding by steady state kinetic methods.
UO
UOInhibition of
E42QMetK
was found to be
also a tight-binding inhibitor of the mutant E42QMetK. The K
was determined by
plotting velocity versus enzyme concentration at fixed
concentrations of substrates and inhibitor, as described previously for
wild type AdoMet synthetase. The fit of the data to yielded a value for K
= 0.38 ±
0.06 µM, which is comparable to the value for wild type;
similar to the case with the wild type, enzyme only
15% of the
total uranyl was inhibitory. However, unlike wild type enzyme the
UO
inhibition of E42QMetK could not be
reversed by KCl (up to 100 mM). These results may indicate
that the amide ligand in E42QMetK is an adequate surrogate for the
carboxylate ligand for UO
but not for
K
. An amide ligand (glutamine 11) to
UO
was observed in the crystal structure
of cytochrome b
(27, 41) . A carboxylate
group appears to be a common constituent of K
binding
sites, since it has been observed in all three known structures of
K
binding sites in
enzymes
(7, 8) .
binding site in
the x-ray structure of AdoMet synthetase was likely to be the
K
binding site. Since glutamate 42 was the only
negatively charged residue at this site, the objective was to
selectively disrupt the binding site by altering the negatively charged
glutamate to the neutral, and nearly isosteric, glutamine residue.
Analysis of E42QMetK revealed a mutant enzyme which maintained the
structural integrity of the wild type AdoMet synthetase, yet displayed
kinetic constants and activity that were nearly identical to that of
wild type enzyme deprived of a monovalent cation activator. These
results indicated that glutamate 42 is important for monovalent cation
activation.
. Additionally, unlike lanthanide ions, which often
form inhibitory complexes with ATP, free UO
rather than UO
ATP is the
predominant inhibitory species. UO
could
thus be designated as a K
antagonist. In a fashion
similar to the activation by K
,
UO
inhibition predominantly affected the
AdoMet formation step and not the PPP
hydrolysis reaction.
UO
inhibits hydrolysis of the ATP analog
A(S)TP, which reinforces the notion that the K
site is
involved in productive binding of ATP. All of the results are
consistent with glutamate 42 being a ligand to K
and
essential for monovalent cation activation.
activation, the fact that
UO
inhibits E42QMetK shows that glutamate
42 is not essential for UO
binding.
UO
binds to amines, amides, and ketones,
as well as carboxylic acids(28) . UO
binds
10
-fold tighter than K
to
the wild type enzyme and may be able to reorder the metal binding
pocket in the E42Q mutant, whereas K
cannot.
Similarly, Falke and co-workers (29) have found mutations in
the Ca
binding site of the E.coli galactose-binding protein that cause up to a four order magnitude
decrease in the affinity for Ca
without detrimental
effects on the affinity for certain trivalent lanthanide ions.
Alternatively, a different ligation geometry may be induced upon
UO
binding in the monovalent cation site.
This postulate is supported by the fact that the central uranium ion of
UO
is significantly smaller than
K
(cation radius of U(VI) = 0.87 Å versus K(I) = 1.52 Å) and uranyl binds more
tightly than K
. Enzymes are known to minimize vacant
space by changing ligation patterns when binding a smaller
metal(7, 30) . The perturbed structure may in fact
give rise to the inhibition of the wild type enzyme. This proposal is
analogous to the mechanism of Na
deactivation of
K
activated dialkylglycine decarboxylase(7) .
The smaller Na
ion induces a different ligation
pattern when it binds to dialkylglycine decarboxylase, which in turn
translates into adverse alterations in the structure of the active
site. The fact that K
is an activator of AdoMet
synthetase and UO
binds at the same site
and completely inhibits seems to indicate that
UO
locks the enzyme in a unproductive
conformation, while K
promotes a productive
conformation. The absence of a monovalent cation may leave the enzyme
free to interconvert between the two states.
binding site as opposed
to the Mg
site warrants some consideration. A
literature survey summarized in Table 3indicates the U(VI) ion
is comparable in size to Mg
(0.86 Å). However,
the average metal-to-oxygen (M
O) ligand bond distance of
uranyl (2.48 Å) is much larger than that of magnesium (2.04
Å), which may explain why the uranyl cation prefers to bind at
the K
rather than the Mg
site of
AdoMet synthetase. Furthermore, like K
,
UO
may have more than six ligands.
UO
binds
10
-fold tighter
than K
, probably due to the additional positive
charge. M
metal ions have been shown to bind at
divalent metal binding sites in enzymes typically with binding
constants 10
-fold tighter than their M
counterparts (31) . The tight-binding inhibition by
UO
is a novel observation, which may be
useful in future studies. The uranyl cation is spectroscopically
versatile having highly characteristic UV and fluorescence spectra (23, 24) and is photo-reactive, generating free
radicals that may cleave the peptide
backbone(17, 18, 36, 37, 38, 39, 40) .
Combined with the possibility of very tight-binding, uranyl may be
useful as a probe to study other K
-activated proteins.
binding site of AdoMet synthetase and those in the
two other known structures of K
-activated enzymes,
pyruvate kinase and dialkylglycine
decarboxylase(7, 8) . In all three enzymes, the
K
binding site is close to or in the active site, but
K
does not interact directly with any substrates or
products. The binding pocket contains one carboxylate residue. The
structure of the K
binding pocket in dialkylglycine
decarboxylase depends on the size of the bound monovalent cation.
Changes in the K
binding pocket structure translate to
changes in the structure of the active site, thereby affecting activity (7) . The K
binding sites of pyruvate kinase (8) and AdoMet synthetase
reveal an even more
notable similarity. The carboxylate ligand to the K
in
each case is also hydrogen-bonded to an arginine residue, which in
pyruvate kinase forms a hydrogen bond to the P
of ATP. Therefore,
in both pyruvate kinase and AdoMet synthetase, the K
may be linked to the ATP substrate via an intervening
carboxylate-arginine amino acid bridge. As Reed and co-workers pointed
out, the carboxylate-arginine bridge provides a path by which cation
size-dependent changes in the monovalent cation binding pocket can be
readily translated to changes in the active site structure and
activity. Whether this turns out to be a common motif in the structural
basis for monovalent cation activation awaits further investigation.
,
tripolyphosphate; PP
, pyrophosphate; P
,
inorganic phosphate; IPTG,
isopropyl-
-D-thiogalactopyranoside;
UO
(Ac)
, uranyl acetate; A(S)TP,
5`-mercapto-5`-deoxy-ATP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.