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INTRODUCTION |
The expression of the pyrimidine biosynthetic (pyr)
operon in Bacillus subtilis and many other bacteria is
regulated by a pyr mRNA-binding protein called PyrR (for
review, see Ref. 1). PyrR acts by binding to a specific RNA sequence
and secondary structure in the attenuation regions of pyr
operons or genes (2). Binding of PyrR at this site stabilizes a
secondary structure in the mRNA that favors formation of a
downstream intrinsic transcription terminator RNA stem-loop, which
results in reduced expression of the full-length mRNA that encodes
the pyrimidine biosynthetic genes. Because the binding of PyrR to its
specific sites is enhanced by uridine nucleotides UMP and UTP (2), this
mechanism leads to repression of pyr gene expression by
pyrimidines in the growth medium. Our laboratory has characterized RNA
binding by PyrR in detail (2), identified amino acid residues in the
RNA binding site of PyrR (3), and characterized the mechanism of
transcription attenuation in B. subtilis (4-6).
A remarkable property of PyrR, first discovered by Ghim and Neuhard in
studies of PyrR from the thermophile Bacillus caldolyticus (7), is that it also catalyzes the uracil phosphoribosyltransferase (UPRTase)1 reaction. This
finding was unexpected because the deduced amino acid sequences of PyrR
proteins from various bacteria bear no significant sequence similarity
outside of a short sequence in the active site to the sequences of
previously characterized bacterial UPRTases, which are encoded by
upp genes (1). Nonetheless, purified B. subtilis PyrR, when assayed under optimal conditions, has a
UPRTase-specific activity comparable with purified bacterial upp-encoded UPRTases (8). Furthermore, the three-dimensional structure of B. subtilis PyrR, which was solved at high
resolution by Tomchick et al. (9), demonstrated that the
PyrR structure is very similar to other Type I
phosphoribosyltransferases (10). We have speculated that PyrR arose
from evolution of an ancestral phosphoribosyltransferase, possibly a
hypoxanthine guanine phosphoribosyltransferase, which PyrR most
resembles in sequence and tertiary structure, by gaining the ability to
bind to a specific RNA structure (9)
This communication presents a kinetic study of the UPRTase
reaction catalyzed by PyrR. We have sought to determine the
relationship between this reaction and the UPRTase reaction catalyzed
by upp-encoded UPRTases and by Type I
phosphoribosyltransferases in general. Our results indicate that PyrR
is a rather typical phosphoribosyltransferase. We propose a kinetic
model for the UPRTase reaction that explains how it displays a Ping
Pong steady state kinetic pattern but does not function via a
phosphoribosyl-enzyme intermediate. Our model provides a mechanistic
resolution to a number of apparently contradictory kinetic studies of
other phosphoribosyltransferases in the literature. Some observations
on the relationship between the UPRTase activity of PyrR and its
function as an mRNA binding attenuation regulatory protein are also presented.
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EXPERIMENTAL PROCEDURES |
All procedures are described in greater detail in Grabner
(11).
Materials--
B. subtilis PyrR was purified by the
procedure of Turner et al. (8) with the following
modifications. The supernatant fluid from the streptomycin
precipitation step was applied directly to the QAE-Sepharose column
without ammonium sulfate precipitation or dialysis, the column was
washed with 400 ml of Buffer R (100 mM Tris acetate,
10 mM Na+-phosphate, pH 7.0) (8) containing 150 mM NaCl instead of 100 mM NaCl, and PyrR was
eluted with an 800-ml linear gradient from 150 to 250 mM
NaCl in Buffer R. The purified PyrR fractions were concentrated with
powdered ammonium sulfate to 80% saturation and dialyzed three times
against 2 liters of 100 mM Tris acetate, 10 mM
potassium acetate, 10% glycerol, pH 7.5, at 4 °C.
PRPP, uracil, UMP, UDP, and UTP were commercial products of
Sigma-Aldrich. The concentrations of PRPP solutions was determined after preparation and monthly thereafter by a variation of the forward
UPRTase assay using limiting PRPP, excess [14C]uracil,
and PyrR and allowing the reaction to proceed to completion. The
concentrations of solutions of uracil and uridine nucleotides were
determined spectrophotometrically.
[
-32P]UTP (25 Ci/mmol) was the commercial product of
ICN Biomedicals, and [14C]uracil (50 mCi/mmol) and
[32P]PPi (1000 mCi/mmol) were from
PerkinElmer Life Sciences. [32P]UMP was prepared from
commercial [
-32P]UTP by hydrolysis in 0.25 N HCl at 100 °C for 35 min. The radiochemical purity of
the [32P]UMP was determined to be 71% by thin layer
chromatographic analysis on polyethyleneimine-cellulose plates.
Impurities were UDP (2%), Pi (11%), and unknown (16%).
The [32P]UMP was diluted with pure nonradioactive UMP of
known concentration for use in equilibrium dialysis, correcting the
specific radioactivity for its 71% purity. [32P]PRPP was
synthesized from 10 nmol of ribose 5-phosphate and 10 nmol of
[
-32P]ATP (5 µCi/nmol, ICN Biochemicals) using 10 µg (0.36 units) of purified human PRPP synthetase isozyme II (12) in
50 mM KH2PO4, 50 mM
triethanolamine, 0.1 mM MgCl2 (in future
experiments 2-5 mM MgCl2 is recommended), pH
8.0, in 100 µl final volume. After reaction for 30 min at room
temperature, unreacted [
-32P]ATP was removed from the
reaction mixture by the addition of 30 ml of a 50% suspension of
acid-washed (13) and neutralized (pH 7) charcoal and incubation on ice
for 30 min followed by centrifugation. The radiochemical purity of the
[32P]PRPP was determined by thin layer chromatography on
polyethyleneimine-cellulose to be 79%; the major impurity was
tentatively identified as [32P]PPi. The
[32P]PRPP was diluted with nonradioactive PRPP of known
concentration for use in equilibrium dialysis, correcting the specific
radioactivity for its 79% purity.
5'-CCUUUUUAAGGGCAAUCCAGAGAGGUUGCAAAGAGG-3', an oligoribonucleotide
predicted from the studies of Bonner et al. (2) to bind to
PyrR with a dissociation constant in the 1-10 nM range was chemically synthesized by Dharmacon Research (Lafayette, CO). Before
its use in equilibrium dialysis the RNA was deprotected according to
the manufacturer's protocol. The RNA concentration of stock solutions
was determined from their UV absorbance.
Assays for UPRTase and Exchange Reactions--
The UPRTase
forward (uracil + PRPP
PPi + UMP) reaction was assayed
by Method 2 previously described (8). The [14C]uracil-UMP
exchange reaction was assayed by the same method, except that PRPP was
omitted from the reactions. The UPRTase reverse (UMP + PPi
uracil + PRPP) reaction was assayed by enzymatic conversion of PRPP
to [14C]AMP with an excess of [14C]adenine
and purified human erythrocyte adenine phosphoribosyltransferase (a gift of Prof. Michael Becker, University of Chicago). Each assay
contained in 50 µl of final volume 0.1 mM
[14C]adenine (14 µCi, ICN Biomedicals), 0.006 IU of
adenine phosphoribosyltransferase (134 µg), 2.5 mM
MgCl2, 50 mM serine buffer, pH 8.7, 30-240
µM UMP, and 300-1000 µM PPi.
It was necessary to incubate all components except PPi at
37 °C for 5 min and to initiate the reactions with PPi
to obtain reaction rates that were linear with the amount of PyrR.
Samples (5 µl) were removed at various time intervals less than 2 min
(necessary to ensure linearity of product formation with time) and
spotted and dried on DEAE-cellulose paper, which was washed to remove
unreacted [14C]adenine and analyzed as for the UPRTase
forward reaction.
The [32P]PPi-PRPP exchange reaction was
assayed by incubating 2 mM
[32P]PPi (8 × 105 cpm), 1 mM PRPP, 5 mM MgCl2, 50 mM serine buffer, pH 8.7, and up to 244 µg of PyrR at
37 °C for 1 h. Uracil, when added, was at 1 mM.
Samples (5 µl) were removed, spotted together with nonradioactive PRPP and PPi, and analyzed by thin layer chromatography
polyethyleneimine-cellulose plates (Selecto Scientific) using 2 M LiCl, 0.75 M ammonium formate, pH 5, as the
developing solvent (28). Authentic [32P]PPi
and [32P]PRPP were also analyzed as standards. In all
cases only one radioactive spot, which co-migrated with
[32P]PPi, was detected, and all of the
radioactivity added to the reaction mixtures was recovered in that spot.
Equilibrium Dialysis Studies of Ligand Binding by PyrR--
The
procedure described by Gibson et al. (14) was used for
equilibrium dialysis, except that ultrathin regenerated cellulose circular (1 cm diameter) dialysis membranes (10,000 molecular weight
cut-off, The Nest Group, Southborough, MA) were used. Dialysis was in
100 mM Tris acetate, 10 mM potassium acetate,
20% glycerol, pH 7.5, at 0 °C for 40-48 h. Equimolar
MgCl2 was included when uracil, UMP, and UTP are ligands;
3- and 2-fold molar excess MgCl2 were added with PRPP and
PPi, respectively. The concentration of PyrR was 100 µM. All experiments were performed in duplicate except
that the ligand and PyrR were mixed together in one chamber and buffer
only was placed in the other chamber ("dialyzing out") in one set,
and the ligand and PyrR were initially in different chambers in the
other set ("dialyzing in"). Coincidence of the two final binding
curves established that equilibrium had been reached. Quadruplicate
samples were removed from the ligand, and PyrR chambers and their
radioactivity were determined by liquid scintillation counting. These
determinations permitted the concentrations of free and bound ligand
and their associated standard errors to be calculated. The data were
fit to a hyperbolic binding curve using Kaleidagraph 3.0 for Windows.
Analysis of Kinetic Data--
Steady state kinetic data were
analyzed using the computer program Kinsim (kindly provided by Bryce
Plapp, University of Iowa), in which the Fortran code of Cleland (15)
was adapted to run on an IBM PC. Bisubstrate saturation kinetics were
fit to the equations for Ping Pong mechanisms to calculate kinetic
constants, which always gave much lower standard errors for the fit
than when the data were fit to the equation for a Sequential mechanism. Product inhibition data were fit to the equations for competitive, noncompetitive, and uncompetitive modes of inhibition. The equation that gave the lowest standard errors was used to determine kinetic constants so long as the pattern of double reciprocal plots was also
clearly consistent with that equation. Steady state kinetic equations
for the four mechanisms shown in Fig. 2, including their product
inhibition patterns, were derived using the method of King and Altman
(17). The details of these lengthy derivations are shown in Grabner
(11).
 |
RESULTS |
PyrR-catalyzed UPRTase Displays a Ping Pong Kinetic Pattern, but
the Enzyme Does Not Catalyze Exchange Reactions Predicted by Formation
of a Phosphoribosyl-Protein Intermediate--
Double reciprocal plots
of the steady state kinetic data for PyrR-catalyzed formation of UMP
from uracil and PRPP were consistently sets of parallel lines (an
example is shown in Fig. 1). This was the
case throughout the pH range from 7.2 to 9.7 and at all substrate concentrations studied. Values for the maximal velocity and Michaelis constants for PRPP and uracil from pH 7.7 to 9.7 have been previously published (8). A Ping Pong kinetic pattern is characteristic of enzymes
that form a covalent intermediate with a portion of a substrate
molecule, which in the case of UPRTase would indicate the following
mechanism.

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Fig. 1.
Double reciprocal plots of substrate
saturation kinetic data for the forward UPRTase reaction in Tris
acetate buffer, pH 8.7. Units = µmol/min. Top,
uracil was the variable substrate with PRPP at 50 ( ), 75 ( ), 150 ( ), 225 (×), and 300 (+) µM. Bottom, PRPP
was the variable substrate with uracil at 50 ( ), 100 ( ), 175 ( ), and 400 (×) µM. Fit of the data shown to the
equation for a Ping Pong mechanism yielded a maximal velocity of
15.3 ± 1.0 µmol/min/mg and Michaelis constants of 59 ± 10 and 171 ± 19 µM for PRPP and uracil,
respectively.
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(Eq. 1)
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(Eq. 2)
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Such a mechanism predicts the ability of the enzyme to catalyze
two independent exchange reactions at rates equal to or greater than
the overall reaction rate, namely [14C]uracil-UMP
exchange and [32P]PPi-PRPP exchange. We
examined the ability of PyrR to catalyze these reactions using the
methods described under "Experimental Procedures." Neither exchange
reaction was detected. No exchange of [14C]uracil into
UMP could be detected using up to 162 µg of PyrR per reaction; in
control experiments in which 100 mM PRPP was included in
the reaction mixture [14C]uracil was incorporated into
UMP at a readily detectable rate (0.024 nmol/min) with only 0.0162 µg
of PyrR added. In attempts to observe
[32P]PPi-PRPP exchange up to 244 µg of PyrR
was added to each reaction mixture, but no exchange of
[32P]PPi into PRPP was observed. However, in
this case formation of [32P]PRPP from
[32P]PPi and uracil was also not detectable,
so direct proof that the PyrR was active was not obtained. However,
PyrR treated in the same manner was highly active in assays of the
forward UPRTase reaction. As will be shown below, the catalysis of the
reverse reaction by PyrR is exceedingly slow; we believe this accounts for the failure to observe it under conditions used in the
[32P]PPi-PRPP exchange assay.
An Ordered Bi Bi Kinetic Model with a Kinetically Irreversible
Conformational Change Can Account for the Kinetic Behavior of
PyrR-catalyzed UPRTase--
The cardinal property of Ping Pong
kinetics that accounts for the parallel lines observed in double
reciprocal plots is not the formation of a covalent enzyme intermediate
but the imposition of a kinetically irreversible step between the
binding of the first substrate and the binding of the second substrate.
In the mechanism shown above that irreversible step is the dissociation of the first product, PPi, into a solution in which its
initial concentration is zero. We therefore considered whether one of several Ordered Bi Bi kinetic mechanisms in which a kinetically irreversible conformational change occurs during catalysis could account for our kinetic observations. In devising these models we took
into consideration the fact that several Type I
phosphoribosyltransferases have been experimentally demonstrated to
undergo large conformational changes after the binding of PRPP (16). We
reasoned that if such a conformational change occurred in PyrR after
the binding of PRPP and this step were kinetically irreversible, such a
mechanism would give rise to Ping Pong kinetic behavior (by
"kinetically irreversible" we mean that the forward direction is
very much faster than the reverse direction and that the reverse
reaction, if it occurs at all, is very much slower than the other
kinetic steps of the mechanism.) This presumption was verified by
derivation of the steady state rate equations for the four kinetic
mechanisms shown in Fig. 2 by the method
of King and Altman (17). These four mechanisms are all Ordered Bi Bi
mechanisms in which PRPP is the first substrate to bind and UMP is the
last product to dissociate from the enzyme. In all cases the
kinetically irreversible step is postulated to be a conformational
change that occurs after the binding of PRPP but before the binding of
uracil. The mechanisms differ in the nature of the step at which the
enzyme returns to the unbound conformation. The steady state equation
for the reaction in the absence of products for all four mechanisms is
the same and is that found for any bisubstrate Sequential kinetic
mechanism.

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Fig. 2.
King-Altman diagrams for the four kinetic
mechanisms for the UPRTase reaction discussed in the text.
E, enzyme; E', enzyme after a conformational
change that follows PRPP binding; A, PRPP; B,
uracil; P, PPi; Q, UMP. Rate
constants for steps in the forward reaction are shown outside the
figure; rate constants for the reverse reaction are shown with minus
signs inside the figure. The four mechanisms differ only in the step at
which the enzyme returns to the unliganded conformation.
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(Eq. 3)
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However, if one assumes that the rate constant for the reverse of
the conformational change, k
2, approaches
zero, the equation simplifies to that obtained for Ping Pong
mechanisms.
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(Eq. 4)
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Thus, all four mechanisms predict the absence of a slope effect in
double reciprocal plots. However, if the product terms are
included, the four mechanisms differ in the product inhibition patterns they predict (Table I). Complete
details of the derivations and deduction of kinetic patterns are
presented in Grabner (11).
Product Inhibition Studies Favor Model 1--
Product
inhibition studies of PyrR-catalyzed forward reaction (PRPP + uracil
UMP + PPi) were conducted and are summarized in Table
II. The pattern of inhibition could be
unambiguously assigned on the basis of the quality of the statistical
fit of the data to the appropriate equations in all cases except for inhibition by PPi as a function of uracil concentration. In
that case uncompetitive inhibition could be excluded, but the fit of the data to competitive and to noncompetitive patterns was essentially equivalent. Table I lists the patterns of product inhibition predicted
for Ping Pong, Random Bi Bi, Ordered Bi Bi (18), and the four
irreversible conformational change models described in Fig. 2. It can
be seen that, even with ambiguity in the pattern of PPi
inhibition versus uracil saturation, our observations are consistent only with Model 1 of Fig. 2.
Also shown in Table II are the results of inhibition studies performed
with UDP and UTP. These nucleotides were investigated because it has
been shown that they, like UMP, affect the affinity of PyrR for
pyr mRNA (2) and stimulate termination of transcription by PyrR in vitro (6). The patterns of inhibition of
PyrR-catalyzed UPRTase by UDP and UTP were the same as for UMP
inhibition, but the inhibition constants were about 2- and 8-fold
larger, respectively. These inhibition patterns suggest that all three
uridine nucleotides bind to the free enzyme and that they all bind to
the UPRTase active site.
The PyrR-catalyzed Reverse UPRTase Reaction (UMP + PPi
PRPP + Uracil) Is Extremely Slow--
We measured
the rate of the reverse reaction by conversion of PRPP to AMP with an
excess of [14C]adenine and adenine
phosphoribosyltransferase as described under "Experimental
Procedures" and found that this rate was more than 3 orders of
magnitude slower than the forward reaction. Indeed, the results of our
attempts to observe the reverse reaction in the context of the
[32P]PPi-PRPP exchange studies indicated that
it could not be detected at all without the use of the adenine
phosphoribosyltransferase coupling enzyme to trap PRPP as quickly as it
is formed. Detailed kinetic studies of the reverse reaction were made
very difficult by its slow rate and two other factors. Very high
concentrations of PPi were required, which caused formation
of precipitates with Mg2+ under the assay conditions. Also,
we observed that the reverse reaction ceased when only a small
percentage of the substrates was consumed. The amount of products
formed was severalfold greater than and did not depend on the
concentration of PyrR, so the reverse reaction was not limited to a
single "burst." Despite these difficulties, we were able to conduct
a study of the dependence of the rate of the reverse reaction on
substrate concentration under conditions where the reactions could be
shown to be linear with both time and the amount of PyrR used. The data
fit best to the equation for a Ping Pong mechanism and yielded values
of 0.0045 ± 0.001 µmol/min/mg for Vmax
(kcat = 1.5 ± 0.3 × 10
3 s
1), 130 ± 37 µM
for the Km for UMP, and 1000 ± 390 µM for the Km for PPi. By
contrast, the kcat for the forward reaction at
the same pH (8.7) was 5.1 s
1, 3300 times higher than for
the reverse reaction.
Studies of the Binding of Substrates to PyrR by Equilibrium
Dialysis--
A prediction of the Ordered Bi Bi kinetic model we have
proposed that accounts for the Ping Pong kinetics we observed is that only the first substrate to bind, PRPP, and the last product to dissociate, UMP, should bind to the free enzyme. We were also interested in examining the binding of these substrates and of UTP, all
of which have been shown to affect the apparent affinity of PyrR for
RNA (2). Binding of commercial [
-32P]UTP,
[14C]uracil, and 32PPi as well as
[32P]UMP and [32P]PRPP, synthesized as
described under "Experimental Procedures," to highly purified PyrR
was studied by equilibrium dialysis. Neither [14C]uracil
nor 32PPi bound detectably to 100 µM PyrR at an initial concentration as high as 1.5 mM, but [32P]UMP and [32P]PRPP
bound well (Fig. 3, A and
B). [32P]UMP binding was well described by a
simple hyperbolic curve corresponding to a dissociation constant of
27 ± 3.2 µM. [32P]PRPP binding was
also hyperbolic and fit a dissociation constant of 18 ± 2.4 µM. Binding at saturating ligand extrapolated to
0.56 ± 0.02 mol of UMP per mol of PyrR subunit and 0.79 ± 0.03 mol of PRPP per mol of PyrR. This stoichiometry of binding varied somewhat from experiment to experiment but was generally less than 1 mol of ligand per mol of PyrR. The reason for this is not known. The
UPRTase activity of PyrR was shown to be constant over the time
required for dialysis. Molecular sieving analysis by high performance
liquid chromatography indicates that PyrR is present entirely as a
hexamer at the concentration used for equilibrium dialysis (3),
although it is possible that some more highly aggregated forms that
fail to bind substrates also form during the 40-48-h dialysis. Binding
of PRPP and UMP to the free enzyme and failure of uracil and
PPi to bind are consistent with the predictions of an
Ordered Bi Bi mechanism in which PRPP binds first and UMP dissociates
last and with each of the four models described in Fig. 2. Such binding
is inconsistent with a Ping Pong mechanism or a Random Sequential
mechanism.

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Fig. 3.
Binding of substrates and UTP to PyrR, as
determined by equilibrium dialysis. A, binding of
[32P]PRPP. B, binding of
[32P]UMP. C, binding of
[ -32P]UTP. In all experiments data from dialyzing in
and dialyzing out protocols were combined. In panel C the
data from two binding studies were combined.
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Binding of [
-32P]UTP was too weak to be well
characterized by our equilibrium dialysis conditions (Fig.
3C). Binding of UTP was clearly above background, but the
dissociation constant could only be characterized as greater than 800 µM.
Equilibrium dialysis studies of the binding of [32P]UMP
and [
-32P]UTP were repeated with 100 µM
each of PyrR and a 36-nucleotide pyr RNA that was known from
the studies of Bonner et al. (2) to bind to dilute solutions
of PyrR with a dissociation constant in the nanomolar range. The
presence of the RNA had no statistically significant effect on the
amount of either nucleotide bound to PyrR or their dissociation constants.
 |
DISCUSSION |
Kinetic Mechanism of the PyrR-catalyzed UPRTase
Reaction--
Kinetic and binding constants for the PyrR-catalyzed
UPRTase reaction are summarized in Table
III. Although we were not able to conduct
a product inhibition study of the reverse reaction and, thus, determine
a complete set of steady state kinetic constants, the results allow a
mechanism to be suggested. We propose here a kinetic mechanism for
the UPRTase reaction catalyzed by B. subtilis PyrR that
is basically an Ordered Bi Bi mechanism with PRPP binding first and UMP
dissociating last. However, to account for the Ping Pong kinetic
patterns consistently observed with this enzyme, we have postulated in
addition a kinetically irreversible conformational change following the
binding of PRPP. This postulate was prompted by the abundant evidence
in the literature for conformational changes that accompany or follow
binding of PRPP to several Type I PRTases (16, 19-22). Model 1, which
fits the observed patterns of product inhibition best, also postulates
that PyrR returns to the unliganded conformation before the
dissociation of UMP. The following experimental observations are
consistent with the proposed kinetic mechanism. (a) Exchange
reactions predicted by a classical Ping Pong mechanism involving
formation of a phosphoribosyl-enzyme intermediate were not detected.
(b) The reverse of the PyrR-catalyzed UPRTase reaction was
extremely slow, 3300 times slower than the forward reaction. This
observation is consistent with a very slow reversal of the
E'(PRPP) conformation to the E(PRPP)
conformation, but of course it does not specifically identify this step
as rate-limiting for the reverse reaction. (c) PRPP and UMP
bound with high affinity to free PyrR, but uracil and PPi
did not bind at all at concentrations up to 0.8 mM. This
result is consistent with an Ordered Bi Bi mechanism. (d)
The kinetic patterns of product inhibition were inconsistent with all
of the other mechanisms considered (Table I). (e) The
experimentally determined value for the equilibrium constant for the
UPRTase reaction, which is 1.2 to 1.5 × 104 at pH 8.5 and 37 °C (29), agreed within a factor of 2 with the value
calculated from the Haldane relationship for our proposed mechanism,
|
(Eq. 5)
|
We consider this agreement to be acceptable given that the value
for the Michaelis constant for PPi could not be determined very accurately and that the dissociation constants for binding of PRPP
and UMP used for estimates of the corresponding inhibition constants
were determined at pH 7.5 and 0 °C. As is always true of steady
state kinetic analysis, consistency of the observations with a given
kinetic model does not establish that model conclusively. A complete
study of the PyrR-catalyzed UPRTase reaction using pre-steady state
methods would be needed to test our model further.
Implications for the Mechanism of Other PRTases--
The Type I
phosphoribosyltransferases all catalyze the transfer of a
phosphoribosyl group from PRPP to a nitrogenous nucleophilic acceptor
with inversion of configuration about C-1 of the ribose moiety, and
they all share highly homologous three-dimensional structures (16). It
is very likely that the mechanism of catalysis of these PRTases is
fundamentally the same. However, both Ping Pong (23-27) and Sequential
(28-31) steady state kinetic patterns have been frequently reported.
The human HGPRTase has even been reported to display Ping Pong kinetics
under some experimental conditions and Sequential kinetics under others
(32). A classical Ping Pong mechanism predicts the existence of a free
phosphoribosyl-enzyme intermediate and capacity to catalyze exchange
reactions between reactant pairs in the absence of co-substrates. These
properties have been reported for various PRTases (27, 33), but on
further scrutiny the conclusions were invalidated (28, 34, 35). In no
case has persuasive evidence for a classic Ping Pong mechanism been put
forward for a Type I PRTase. How, then, are the frequent Ping Pong
kinetic patterns to be explained? We believe that our irreversible
conformational change model for PyrR-catalyzed UPRTase can provide a
general solution to this question.
We cited evidence above from both structural and kinetic studies that
some PRTases undergo large conformational changes upon the binding of
PRPP. In the case of Escherichia coli glutamine PRPP
amidotransferase this conformational change was shown to be relatively
slow (19). In a very detailed kinetic study, Xu et al. (31)
identify the product release step of human HGPRTase, which follows an
Order Bi Bi kinetic mechanism, to be rate-limiting in both directions.
They suggested that these steps were slow because of rate-limiting
conformational changes that accompany or follow them. We propose that
binding of PRPP to the free enzyme followed by a conformational change
and then by binding of the second substrate is a general property of
the Type I PRTases. Furthermore, we suggest that the reversibility of
this conformational change determines whether the enzyme will display a
Sequential or a Ping Pong kinetic pattern, with the latter resulting
from very slow reversal. If this view is correct, we would predict that
PRTases displaying Ping Pong kinetic patterns will be either kinetically irreversible or show much slower rates of their reverse reaction than of the forward (PRPP-dependent) reaction. The
ratio of catalytic rates constant for the forward versus for
the reverse reaction is about 100 for upp-encoded UPRTase
from E. coli, for example, which follows a Sequential
kinetic pattern (29). Thus, we propose that this ratio would be
1000-fold or greater for PRTases that obey Ping Pong kinetics. This
prediction is relatively easy to test. Note that it is not necessary
that a given PRTase displaying Ping Pong kinetics obey Model 1; many
variants can be imagined that predict the same pattern.
Implications Concerning the Physiological Functions of
PyrR--
Even though it bears little amino acid sequence similarity
to the UPRTases encoded by bacterial upp genes, PyrR has
catalytic properties that are typical of most PRTases. The only
significant exception to this was reported previously (8): PyrR
has Km values for uracil at physiological pH values
that are more than 2 orders of magnitude larger than observed with
bacterial UPRTases. Thus, PyrR would be relatively ineffective in
uracil salvage. However, B. subtilis (36) and B. caldolyticus (7) pyrR genes have been shown to
encode functional UPRTases in vivo.
Two of the most important effectors of PyrR binding to pyr
mRNA, UMP and PRPP, are a product and a substrate, respectively, of
its UPRTase activity. Our results are consistent with the proposal that
these molecules affect PyrR binding to RNA by binding to the UPRTase
active site, although they cannot conclusively prove this. The
dissociation constants for UMP and PRPP are reasonably close to the
concentrations at which they exert half-maximal effects on RNA binding
(2) or transcription termination (6), especially given the differences
in reaction conditions used in the different studies. PRPP antagonizes
the effects of UMP on transcription termination (6), which indicates
competition for the same site, a result also obtained in our kinetic
studies. UTP exerts similar effects as UMP on transcription termination
(6) and binding of PyrR to RNA (2), but 10-100-fold higher
concentrations of UTP are required. The kinetic pattern of inhibition
of UPRTase activity by UTP and its 10-fold larger inhibition constant
supports the idea that UTP also regulates the attenuation functions of PyrR by binding to the UMP portion of the UPRTase active site. Binding
of UTP to PyrR was appreciably weaker than we expected, however.
Because UMP and PRPP affect the affinity of PyrR for pyr
mRNA, one might expect a reciprocal effect, i.e.
alteration of binding of UMP or PRPP by RNA in equilibrium dialysis
experiments or effects of RNA on the kinetics of UPRTase activity. No
such effects were observed either in the dialysis experiments or in
studies of UMP and PRPP saturation of PyrR in UPRTase assays at
37 °C (Ref. 11 and data not shown). However, there was no way to
prove that the pyr RNA was bound to PyrR under the
conditions of equilibrium dialysis, where the concentration of PyrR was
much higher than in RNA binding studies, or at the assay temperature of
37 °C (the binding studies were conducted at 0 °C). A more direct
analysis of the interactions between the UPRTase active site and the
RNA binding site of PyrR must await the determination of a high
resolution structure of a PyrR-RNA complex. Attempts to achieve this
goal are in progress.