From the
Equilibrium isotope exchange kinetics (EIEK) and kinetic isotope
effects have been used to determine the mechanistic basis for the
altered kinetic characteristics of a mutant version of Escherichia
coli aspartate transcarbamoylase in which Asp-236 of the catalytic
chain is replaced by alanine (Asp-236
Aspartate transcarbamoylase (EC 2.1.3.2) from Escherichia
coli (ATCase)
One of the major unanswered questions
about the dynamic behavior of ATCase regards the rate-limiting step in
the forward direction. The properties of the Asp-236
[
On-line formulae not verified for accuracy
Two methods were used to determine the isotopic
content of unreacted CP. In the first, CP was converted to CO
This apparent
similarity is not supported by a more detailed comparison, however.
First, considering kinetic isotope effects, the saturation curve for
By comparing how the kinetic
parameters for each of these forms differ from those for wild-type
holoenzyme, a set of distinctive patterns emerges. In order to analyze
these effects systematically, the perturbations in
R
Using these initial clues as a working
hypothesis, simulation of the kinetic data for Asp-236
Disrupting the interchain interaction between Asp-236 of a
catalytic chain and Lys-143 of the regulatory chain destabilizes the
T-state of ATCase
(10) . The current kinetic results, in
conjunction with structural considerations, are consistent with the
mutation causing an increase in k
Of particular significance for discerning
which steps contribute to overall rate-limitation, the maximum activity
for the Asp-236
These and previous findings suggest that binding of Asp to ATCase is
a complex, multistep process. Overall, this involves both gross
quaternary structural changes (the T
A key
feature of the mechanism for Asp cooperativity depicted in Schemes I
and II is ``induction'' of the initial conformational change
caused by the binding of Asp to the low affinity form of ATCase. Based
on x-ray structural data
(35) , binding of CP alone alters the
free T-state enzyme. CP-liganded enzyme is designated T` to distinguish
it from the pure T-state (unligated), which cannot bind Asp. The first
molecule of Asp to bind triggers ( induces) formation of an
activated, highly transient T-state form, designated T*, which rapidly
undergoes concerted conversion of all three chains to a near-R-state
conformation designated R`. This is in accord with the observation that
only one PALA molecule per catalytic trimer results in a complete T
At a more detailed
structural level, Fig. SIIindicates that binding of the first
Asp (in step 1) triggers disruption of specific bonds that stabilize
the T`-state. In the current model, these are suggested to be residues
in the 240s loop such as the intrachain hydrogen bonds and salt links
between Tyr-240-Asp-271 and Arg-229-Glu-272. Binding of the
first Asp produces the labile, activated state T*, the least stable
intermediate in this process, which readily converts its gross,
quaternary structure to R`. This latter form is proposed to have
enhanced affinity for Asp without having achieved an active site
conformation with full catalytic activity. Attaining the fully active
R-state depends on stepwise binding of Asp to the remaining active
sites (in step 2) by mass action. As described above, this drives
completion of domain closure and brings active site groups into proper
juxtaposition, to facilitate the ``compression'' mechanism
(39) in which the carbonyl carbon of CP undergoes nucleophilic
attack by the
Data in the current literature allow us to
eliminate certain steps in Fig. SIIas candidates for the
non-catalytic rate-limiting step, specifically step 1 and the T
CTP, which clearly pushes the T
This paper is
dedicated to the memory of Dr. Frederick C. Wedler, who died on
December 1, 1994. During his career he made important contributions to
the understanding of a variety of enzymes including aspartate
transcarbamoylase using equilibrium isotope exchange kinetics.
We thank Dr. P. Vachette for making available the
results of low angle x-ray experiments prior to publication. We also
thank Dr. S. C. Pastra-Landis and Dr. D. Baker for critically reviewing
this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Ala). The
[
C]Asp
N-carbamyl-L-aspartate (CAsp) and
[
C]CP
CAsp exchange rates, observed as
a function of various reactant-product pairs, exhibited dramatic
increases in maximal rates, along with decreases in substrate
half-saturation values and cooperativity. The carbon kinetic isotope
effect,
C versus
C at the carbonyl
group of carbamoyl phosphate, for the Asp-236
Ala enzyme
decreased toward unity as [Asp] increased, as observed for
the wild-type enzyme. Both the kinetic isotope effects and EIEK results
indicate that the Asp-236
Ala enzyme operates by the same
ordered kinetic mechanism as the wild-type enzyme. Although activation
effects by ATP and N-phosphonacetyl-L-aspartate are
lost, inhibition by CTP was apparent in equilibrium exchanges.
Simulation of the EIEK data indicated that the best fit to the observed
changes in saturation curves was obtained by preferentially increasing
the rate of the T
R transition, k
,
thereby destabilizing the T-state and increasing the equilibrium
constant for the T
R transition. A multistep model for Asp
binding to aspartate transcarbamoylase is proposed, in which Asp
induces the initial conformational changes that in turn
trigger the T
R transition, followed by stepwise filling of the
remaining active sites.
(
)
is the primary regulatory
enzyme of the pyrimidine biosynthetic pathway, exhibiting a positive
homotropic interaction with the substrate L-aspartate, along
with heterotropic activation by ATP and synergistic inhibition by the
end products CTP and UTP
(1, 2, 3) . The
holoenzyme ( M
310,000) is composed of catalytic
(c) and regulatory (r) subunits with overall stoichiometry
c
r
, arranged as
(c
)
(r
)
or two trimers
of catalytic chains ( M
34,000 each) and three
dimers of regulatory chains ( M
17,000 each). The
three-dimensional structure of the enzyme has been determined to high
resolution, and structural differences between the T- (low affinity,
low activity) and R- (high affinity, high activity) states are well
documented
(4, 5, 6, 7) . The T- to
R-state conversion involves major quaternary structural changes,
including a 12-Å expansion along the 3-fold molecular axis, and
rotation of the regulatory subunits about their respective 2-fold axes
(8) , along with more subtle tertiary structural changes. These
include closure of the cleft between the two domains of each catalytic
chain, and adjustments of the structure within the Asp binding site. In
addition, within each catalytic chain, a section composed of residues
230-250 (the 240s loop) undergoes a major reorientation. For the
T-state holoenzyme, specific side chain interactions also occur between
catalytic chains of the two catalytic trimers, and between Asp-236 of
the catalytic chains and Lys-143 of the regulatory chains
(9) .
Analysis of the Asp-236
Ala(
)
enzyme has
revealed the importance of these interactions for both homotropic and
heterotropic interactions in the enzyme
(10) . Initial velocity
kinetics indicated that the mutant holoenzyme lacked cooperativity for
substrate binding, while substrate affinities have increased by
3-10-fold. Most striking was an increase in V
of
25%. Only 2 or 3 of the 50 or more site-specific mutants
of ATCase prepared as of 1990 exhibit such increases in the maximal
velocity
(11) . Mutation of Asp-236
Ala and Tyr-240
Phe
(10, 12) , both in the 240s loop, are almost
unique in causing a significant increase in
V
.
Ala enzyme
make it valuable for gaining new insights toward this question. An
in-depth kinetic analysis is required to define changes in individual
rate constants. Equilibrium isotope exchange kinetics (EIEK) and
kinetic isotope effects (KIE) have proven highly insightful with ATCase
(13, 14, 15, 16, 17, 18, 19) .
Unique to EIEK methods is the ability to observe simultaneously both
the fast and slow steps for substrate association-dissociation in both
directions, involving binary and ternary complexes. For example, it was
found that the allosteric modifiers ATP and CTP do not perturb the T
R transition directly; instead, they differentially alter the
Asp association rate more than the dissociation rate
(20) . For the Tyr-240
Phe mutant, computer simulations
of EIEK data elucidated changes in the kinetic mechanism
(13, 14) . KIE methods have been shown to be sensitive
to alterations in the chemical and kinetic mechanism of the wild-type
and mutant enzymes
(13, 14, 15, 16, 17, 18, 19) .
The present investigation involves using both these methods to
investigate kinetic changes caused by the Asp-236
Ala mutation,
which are relevant for helping to define in greater detail the
rate-limiting step in the forward direction for the wild-type enzyme.
Materials
Biochemicals were of the highest
purity available from Sigma. Other chemicals were ACS reagent grade
(Fisher Scientific). Carbamyl phosphate (dilithium salt) was purified
prior to use by precipitation from 50% (v/v) ethanol:water and was
stored desiccated at 20 °C.
[
C]L-Asp (Amersham Corp., 50 µCi,
216 mCi/mmol) was purified from radiolytic contaminants prior to use by
application of the entire sample to a 0.3
1.0-cm column of
Dowex-50 AG-X8 (H
, 100-200 mesh), followed by a
15-ml water wash, then elution with 10 ml of 2 M HCl. After
taking the eluant to dryness twice by rotary evaporation, the residue
was dissolved in 1 ml of 20% ethanol:water and stored at
20
°C. [
C]Carbamyl phosphate (DuPont NEN, 50
µCi, 17.6 mCi/mmol) was dissolved in 10 ml of water, divided into
100-µl aliquots, flash-frozen with liquid nitrogen, and stored at
80 °C. These aliquots of labeled CP were thawed and used
individually and the residual material was flash-frozen no more than
once to avoid breakdown to cyanate.
Enzymes
Wild-type ATCase was prepared as described
by Nowlan and Kantrowitz
(21) from E. coli strain
EK1104 containing the plasmid pEK2. The Asp-236 Ala ATCase was
prepared as described previously
(10) .
General Methods
Enzyme activity was assayed using
either the formation of [C]CAsp from labeled Asp
(22) , a continuous spectrophotometric coupled phospholysis
method
(23) , or a pH indicator assay.
(
)
One unit of activity is defined as the number of micromoles
of product (CAsp) formed per minute. Protein concentration was
determined by the Lowry method
(24) after trichloroacetic acid
precipitation, by the bicinchoninic acid (BCA) method (Pierce), or by
absorbance measurements at 280 nm with an extinction coefficient of
0.59 cm
/mg
(25) .
Equilibrium Isotope Exchange Kinetics
Equilibrium
isotope exchange experiments were carried out according to established
procedures, described in detail elsewhere
(26, 27, 28) . The concentrations of
substrate-product pairs were varied from well below to well above their
Kvalues as follows; solution A
(containing reaction components at twice their final concentrations)
was mixed in different proportions to the same final volume (0.1 ml)
with solution B (lacking the varied components). After thermal
equilibration for 5 min at 30 °C, 5 µl of enzyme was added and
the mixture was incubated for 15 min to allow for exact catalytic
adjustment to chemical equilibrium. Isotopic exchange was then
initiated by addition of labeled substrate,
[
C]L-Asp (0.1 µCi) or CP (0.02
µCi), in micromolar quantities less than 1/1000 of the material in
the unlabeled pool so as to avoid perturbation of the equilibrium
condition. Exchange reactions were typically carried out for 20 min at
30 °C, then quenched and separated for counting.
C]Asp
C-Asp exchange reactions were
quenched by addition of 0.45 ml of 0.02 M HCl and chilling on
ice prior to separation of the labeled pools, which was carried out
exactly as described above for the purification of
[
C]Asp, using a 0.3
5-cm column of Dowex
50 (H
). The [
C]CP
CAsp
exchange reactions, carried out in 1.5-ml Eppendorf tubes, were
quenched by addition of 0.15 ml of 5 N HCl. The tubes were
closed, the caps pierced, and the [
C]CO
removed in vacuo (<0.5 mm Hg) for 75 min, using a
Savant SpeedVac concentrator with a refrigerated trap plus an Ascarite
(5
20 cm) trap prior to the vacuum source.
Calculations
For exchange of isotopic label at
chemical equilibrium, X* Y, the number of
micromoles exchanged/min ( R) was calculated according to
Equation 1
(29) , where X and Y represent the
micromoles of substrates present in a given reaction, t = time (min), and F is the fraction of isotopic
equilibrium attained, equal to y( X +
Y)/( x + y) Y, where x and y are the disintegrations/min values observed in the
X and Y pools, respectively.
EIEK Data Analysis
The shapes of saturation
curves in EIEK experiments distinguish between compulsory and random
order sequential kinetic mechanisms
(26, 27, 28) . EIEK methods are unique in their
ability to allow simultaneous observation of both the rate-limiting and
non-rate-limiting steps in both directions, which include the formation
and breakdown of both the ``inner'' and ``outer''
complexes. A newly developed mouse-interactive calculational program,
ISOBI-HS, was used to fit entire sets of equilibrium isotope exchange
data
(28) . These procedures lead to an optimal set of rate
constants, as described elsewhere in theory and practice
(13, 14, 20, 28) . The rate constants
must meet several other criteria in order to be considered valid.
Specifically, within determined confidence limits (allowed limits of
variation) they must produce chemical equilibrium for all closed cycles
and predict the correct initial velocities in both directions, using
the KINSIM program
(30) . The kinetic constants and saturation
curves for the mutant are then compared to those for wild-type enzyme,
thereby determining the specific rate constants altered by the
mutation. The theoretical curves in the figures were generated using
the best fit parameters from the ISOBI-HS program
(28) .
Isotope Effects
Isotope effects are described
using the terminology of Northrop
(31) . Thus,
(V/K)
represents the observed kinetic isotope
effect for
C versus
C at the
carbonyl group of CP.
(V/K)
on the ATCase
reaction was measured using the method of internal competition, in
which changes in the isotopic composition of CP are measured over the
course of the reaction
(17) . A 200 mM CP solution, pH
5.5, was prepared in water, sealed with a rubber septum, placed in an
ice bath, and freed from CO
by sparging with N
for 1-2 h. A reaction flask fitted with a vacuum adapter
and a side arm was charged with an appropriate amount of 50 mM
HEPES, pH 7.5, containing 2 mM dithiothreitol and 0.2
mM EDTA, and this solution was sparged overnight with
N
. A 100 mM Asp solution was prepared in this same
buffer, sealed with a septum, and sparged with N
overnight
at room temperature. 50 mM ATP and CTP solutions were prepared
in 50 mM HEPES containing 2 mM dithiothreitol and 0.2
mM EDTA, pH 7.5, and were likewise sparged. To the reaction
flask containing HEPES buffer were added appropriate amounts of the
aspartate solution and ATCase. The reaction was initiated by addition
of 1 ml of ice-cold CP solution. At low Asp concentrations, in order to
keep the Asp concentration constant, small amounts of degassed Asp
solution were added dropwise at a rate calculated to keep the Asp
concentration at the desired level. After a reaction time estimated to
attain 50% reaction, 0.5 ml of concentrated sulfuric acid was added to
the reaction mixture via a syringe and the solution was warmed to 38
°C for 2 h to convert unreacted CP to CO
(>10
half-lives of CP hydrolysis; at pH 0.35, 37 °C, the half-life for
CP is
11.3 min
(32) . The resulting CO
was
collected by a high vacuum continuous distillation apparatus using two
dry ice/isopropanol traps and a liquid nitrogen trap. The trapped
CO
was further purified by bulb to bulb distillations. The
amount of CO
produced was measured manometrically using a
MKS PDR-D1 READOUT pressure gauge calibrated with acidified potassium
carbonate solution. The isotopic content of the resulting CO
was measured by isotope ratio mass spectrometry on a Finnigan
Delta-S isotope-ratio mass spectrometer equipped with a Heraeus
combustion unit.
by acid treatment. 1 ml of sparged CP solution in water was added
to a flask containing 15 ml of HEPES buffer solution. Acidification of
this solution with 0.5 ml of concentrated sulfuric acid resulted, after
2.5 h at 38 °C, in the decomposition of all the CP to give
CO
, which was isolated and analyzed as described above.
Alternatively, combustion analysis was used to obtain the isotopic
content of CP. The two methods gave the same results, within
experimental error.
Equilibrium Isotope Exchange Kinetics
Saturation
curves for the [C]Asp
CAsp and
[
C]CP
CAsp exchanges, catalyzed by the
Asp-236
Ala holoenzyme at pH 7.0, 30 °C, resulting from
variation of both reactants and both products in constant ratio at
equilibrium, are shown in Fig. 1. For comparison, the curves
obtained for wild-type holoenzyme under identical conditions
(29) are indicated by dotted lines. The shapes
of the curves are altered in a manner very similar to the changes
observed by initial velocity kinetics upon variation of the
concentration of Asp
(10) . Specifically, the half-saturation
value decreased markedly, and the maximal rate increased. Whereas
V
increased only about 20%
(10) , the
observed increase in R
for isotope exchange is
increased by 2-fold for Asp
CAsp and by almost 3-fold for CP
CAsp. The strong inhibition effects for CP
CAsp, due
to ordered substrate binding
(10) , are even more apparent for
the Asp-236
Ala holoenzyme (Fig. 1), along with weak
inhibition effects observed previously for Asp
CAsp. These
latter observations suggest that the Asp-236
Ala holoenzyme
operates by a kinetic mechanism essentially identical to that for the
wild-type holoenzyme, namely CP binds prior to Asp and CAsp is released
prior to P
. The data in the next several figures serve to
verify this hypothesis.
Figure 1:
The
effect of varying the concentrations of all substrates in constant
ratio on the rates of the [C]Asp
CAsp
and [
C]CP
CAsp exchange reactions,
catalyzed at chemical equilibrium by the Asp-236
Ala enzyme at
pH 7.0, 30 °C. The maximum concentrations of substrates at f = 1.0 were (in mM): CP (0.185), Asp (185), P
(436), and CAsp (463). Each reaction also contained 100
mM PIPES buffer and 100 mM KCl. Dotted line,
saturation curve for the wild-type enzyme
(30).
Fig. 2
shows the effects on the Asp
CAsp and CP
CAsp exchange rates of varying all
possible reactant/product pairs in constant ratio, catalyzed by the
Asp-236
Ala holoenzyme. Variation of the Asp/CAsp pair
(Fig. 2 A) causes Asp
CAsp to rise smoothly to a
maximum, but results in sharp peaking and inhibition of the CP
CAsp exchange. In contrast, variation of the CP/P
pair
(Fig. 2 B) simply caused both exchanges to rise smoothly
to a maximum, as expected for the preferred order binding mechanism
described above. Weak substrate inhibition effects seen for the
wild-type are absent for the Asp-236
Ala holoenzyme. Strong
enhancement of the maximal rate for the CP
CAsp exchange is
even more apparent in Fig. 2than in Fig. 1, with almost
5-fold increases for the Asp-236
Ala enzyme compared to the
wild-type holoenzyme. In both experiments, both exchanges showed
markedly decreased half-saturation values as well.
Figure 2:
Rates
of the [C]Asp
CAsp and
[
C]CP
CAsp exchange reactions as a
function of the concentrations of reactant-product pairs, varied in
constant ratio at chemical equilibrium, catalyzed by the Asp-236
Ala mutant of ATCase at pH 7.0, 30 °C. Each reaction also contained
100 mM PIPES buffer and 100 mM KCl. The
concentrations of the nonvaried pairs were held constant corresponding
to f = 0.4 in Fig. 1. Variations are as shown:
A, Asp and CAsp, holding [CP] and
[P
] constant at 0.074 and 174 mM,
respectively; B, CP and P
, holding [Asp]
and [CAsp] constant at 74 and 185 mM, respectively;
C, Asp and P
, holding [CP] and
[CAsp] constant at 0.074 and 185 mM, respectively;
D, CP and CAsp, holding [Asp] and [P
] constant at 74 and 174 mM, respectively.
Dotted lines, saturation curves for the wild-type enzyme
(30).
Variation of
Asp/P(Fig. 2 C) results in strong inhibition
of the CP
CAsp exchange rate, with weaker effects on the Asp
CAsp exchange. The fact that weak inhibition of the Asp
CAsp exchange was not observed in Fig. 2 A for
the variation of Asp/CAsp suggests that this effect is due to dead-end
complex formation, perhaps E
P
Asp.
Similar weak inhibition effects are seen in Fig. 2 D upon
variation of CP/CAsp. By similar logic, comparison of these curves to
those in Fig. 2 B suggests that CAsp also forms a
dead-end complex, probably E
CAsp
CP. The data in
Fig. 2
( C and D) clearly show a stronger
increase in maximal rate for the CP
CAsp exchange, compared to
Asp
CAsp. Overall, the data in Fig. 2verify that the
Asp-236
Ala holoenzyme operates by a kinetic mechanism that is
essentially identical to that for the wild-type holoenzyme, namely a
nearly compulsory order scheme in the forward
(17, 29) and reverse
(29) directions. Inhibition effects
that are diagnostic of this scheme are even more distinct with the
Asp-236
Ala enzyme than with the wild-type holoenzyme.
Kinetic Isotope Effects
The C isotope
effect, measured for the reaction of [
C]CP with
Asp, catalyzed by the Asp-236
Ala enzyme as a function of the
concentration of Asp in the presence of a fixed and saturating
concentration of CP, is shown in Fig. 3. The shape of the
saturation curve, defined by the data for the wild-type holoenzyme, is
shown as a dotted line (29) . The data for the
mutant enzyme are identical to those for the wild-type enzyme, within
experimental error.
Figure 3:
Kinetic
isotope effect, (V/K)
, for the forward
reaction catalyzed by the Asp-236
Ala mutant of ATCase as a
function of [Asp] in the presence of fixed [CP] (20
mM), pH 7.5, 25 °C. Each reaction also contained 50
mM HEPES buffer, 2 mMdithiothreitol, and
0.2 mM EDTA. Dotted line, saturation curve for the
wild-type enzyme (17).
The fundamental conclusion derived from the
observed decrease in (V/K)
from
1.025 at
low [Asp] to unity at saturating [Asp] is that the
kinetic mechanism is ordered. If the kinetic mechanism were random, the
isotope effect would remain finite and constant at high
[Asp], since CP could escape from the central complex,
E
CP
Asp. In the case of a compulsory order mechanism,
however, although at low [Asp] bound CP can freely dissociate
and isotopic discrimination occurs, as [Asp] increases bound
CP is increasingly prevented from dissociating by the rapid binding of
the second substrate, Asp (which results immediately in a high
commitment to catalysis). Thus, the observed kinetic isotope effects
are in accord with the ordered kinetic mechanism determined from the
isotope exchange data in Figs. 1 and 2.
Effector Ligands
By initial velocity kinetics, the
Asp-236 Ala enzyme appears to behave as a nearly R-state form
(10) , based on the almost 10-fold decrease in
S
and loss of cooperative binding for Asp, and
simultaneous loss of activation effects by the bisubstrate analog,
PALA. With wild-type holoenzyme under conditions of [Asp]
below S
, low concentrations of PALA (<1
µM) cause activation, due to sub-stoichiometric amounts of
bound PALA converting the low activity, low affinity T-state enzyme to
the high activity, high affinity R-state. Lack of PALA activation
indicates that the Asp-236
Ala enzyme is already in the R-state,
even at saturating CP and low [Asp]. The isotope exchange
data in Fig. 4fail to detect any PALA activation of either Asp
CAsp or CP
CAsp, even at concentrations near 1
µM. Inhibition effects are due to direct competition of
PALA with substrates.
Figure 4:
Effects of varying the concentration of
the bisubstrate analog, PALA, on the rates of the
[C]Asp
CAsp and
[
C]CP
CAsp exchanges at chemical
equilibrium, catalyzed by Asp-236
Ala enzyme at pH 7.0, 30
°C. Substrate concentrations were held constant at concentrations
corresponding to f = 0.15 in Fig.
1.
Another notable feature of the Asp-236
Ala enzyme was loss of sensitivity to modifiers, ATP and CTP under
initial velocity conditions
(10) . Using EIEK methods, however,
these modifiers alter the Asp
CAsp and CP
CAsp
saturation curves (as a function of various reactant/product pairs), as
shown in Fig. 5. The Asp-236
Ala enzyme is insensitive to
ATP (except for weak inhibition of CP
CAsp upon variation of
the CP/P
pair), but in all cases CTP caused strong,
differential inhibition of the CP
CAsp exchange, but not the
Asp
CAsp exchange. Furthermore, CTP alters the
R
but not the half-saturation value for the CP
CAsp exchange, as also occurred with the wild-type enzyme
(20) . A similar differential loss of modifier sensitivity was
observed with the pAR5 enzyme described by Hervé, Cunin, and
co-workers
(33) . Differential changes in modifier sensitivity
can provide important clues about which kinetic steps have been altered
by the mutation, as will be discussed below.
Figure 5:
Effect of
modulators (M) on the exchange rates at chemical equilibrium, catalyzed
by the Asp-236 Ala ATCase enzyme, pH 7.0, 30 °C. Data are
shown for 0.5 mM CTP and 2 mM ATP on the
[
C]Asp
CAsp and
[
C]CP
CAsp exchanges. The
concentrations of substrates, varied together in constant ratio, and
other components were as in Figs. 1-3. Concentrations of
nonvaried components in the two bottom panels corresponded to
f = 0.4 in Fig. 1.
Data Analysis
A useful starting point for
analyzing the effects of the Asp-236 Ala mutation is to consider
the kinetic properties of previously studied forms of ATCase that
exhibit ``activation,'' i.e. increased
V
and decreased S
values.
These enzyme forms include the catalytic subunit of the wild-type
enzyme
(20) , the ATP-ligated holoenzyme
(20) , and two
R-like mutants, Tyr-240
Phe
(13) and Glu-239
Gln
(34) . The properties of these forms are summarized in
. The first impression from this summary is that the
Asp-236
Ala enzyme appears to most closely resemble the
wild-type catalytic subunit in its properties.
(V/K)
for wild-type catalytic subunit does
not go to unity
(17) and therefore maintains a degree of random
character not seen with the Asp-236
Ala or wild-type
holoenzymes, although the data for the Asp-236
Ala enzymes do
not rule out a very small random component to the mechanism. Second,
from equilibrium isotope exchange kinetics, distinctive differences in
the kinetic parameters R
and S
for the Asp
CAsp and CP
CAsp exchanges are
observed for wild-type catalytic subunit and the Asp-236
Ala
holoenzyme; these parameters are listed in , along with
those for the wild-type holoenzyme.
and S
compared to the
wild-type enzyme were calculated and plotted in bar graph form, as
shown in Fig. 6(see ). This method of comparison
has proven highly effective in defining which steps are altered by
bound feedback modifiers
(20) , as well as by site-specific
mutations
(13, 34) .
Figure 6:
Perturbations in EIEK parameters of
``activated'' forms of ATCase, compared to wild-type
holoenzyme, pH 7.0, 30 °C, calculated from data in Table II (see
text). Stippled bar, V(µmol/min/mg); shaded bar),
1/ S
. The enzyme forms are as follows:
A, wild-type catalytic subunits; B, wild-type
ATP-liganded holoenzyme; C, Asp-236
Ala holoenzyme;
D, Tyr-240
Phe holoenzyme; E, Glu-239
Gln holoenzyme.
Although it is clear that the
kinetic behavior of the Asp-236 Ala enzyme is unique, this
comparison indicates a close resemblance of this mutant to both the
wild-type holoenzyme with ATP bound and the Glu-239
Gln enzyme.
Although several other ``activated'' forms exhibit increases
in substrate affinity (1/ S
) values comparable to
those observed for the Asp-236
Ala enzyme, they also exhibit
decreases in R
, whereas the Asp-236
Ala enzyme shows distinctive increases in
R
for both exchanges. The match in perturbation
of parameters (Fig. 6) is best for ATP-ligated wild-type and
Glu-239
Gln enzymes in experiments involving variation of the
concentrations of all substrates or the Asp/CAsp pair. When the
CP/P
pair was varied, only the wild-type holoenzyme with
ATP bound exhibited perturbations that resembled those observed for the
Asp-236
Ala enzyme.
Ala
enzyme was carried out with the ISOBI-HS program. First, the effect of
altering the association and dissociation rates for Asp and CAsp was
determined, both in equal ratio and by differentially increasing
k
> k
. These changes,
as observed previously
(20) , enhanced R
and decreased S
for Asp
CAsp, but
had the opposite effect on the CP
CAsp exchange. This is not
observed with the Asp-236
Ala enzyme, for which both exchanges exhibit increased exchange rate and substrate affinities
(decreased S
). The only changes that produced
the exact effects observed with the Asp-236
Ala enzyme in Figs.
1 and 2 were a decrease in the Hill number ( n
)
from 2.2 to 1.5 plus slightly slower release of Asp from two dead-end
complexes, E
P
Asp and
E
CAsp
Asp.
(but not
k
) for the T
R transition, which
increases the allosteric parameter L
k
/ k
. In accordance with
these findings, low angle x-ray scattering data show that unliganded
Asp-236
Ala enzyme has a quaternary conformation that is neither
T nor R, but that binding of CP alone converts the Asp-236
Ala
enzyme
75% toward the quaternary conformation observed for
wild-type R-state.(
)
The kinetic data in Figs.
1-3 also clearly show that the Asp-236
Ala substitution
does not alter significantly the ordered kinetic mechanism observed for
wild-type holoenzyme.
Ala enzyme is almost twice that of wild-type
enzyme by both EIEK and initial velocity methods.(
)
Comparison of EIEK saturation curves for the Asp-236
Ala
enzyme to those for various ``activated'' forms of ATCase
indicate that the Asp-236
Ala enzyme most closely resembles the
ATP-liganded wild-type enzyme, which has substantial R-state character
(20) . In accordance with this analysis, x-ray crystallographic
data indicate that the binding of ATP specifically disrupts the link
between Asp-236 (catalytic) and Lys-143 (regulatory)
(35) .
R transition) as well as
more subtle secondary structural changes associated with stepwise
binding to the active site once a state with appreciable affinity for
Asp has been achieved. These different levels of structural changes
have been the subject of hypothesis and experimentation by a number of
researchers in recent years, including Tauc et al. (36) . Clearly, the simple two-state M-W-C (concerted)
model
(2) for cooperative substrate binding is an
oversimplification for this system. An attempt to incorporate these
structural changes into a model that relates them to the observed
kinetics and mechanism is outlined in Schemes I and II.
R transition
(37) . The structural basis for expansion of
quaternary structure along the 3-fold axis by 12 Å and movement
of the 240s loops of the catalytic chains past each other has been
described in detail
(38) . Once R` is formed, stepwise filling
of the additional sites, driven by mass action of [Asp],
produces the fully active R-state enzyme.
-amino group of Asp.
Figure SII:
Mechanistic hypothesis for stepwise
substrate binding to ATCase, coupled to gross quaternary (T R)
and more subtle secondary structural conformational
changes.
The kinetic isotope effects
observed by Parmentier et al. (17) indicated that
catalysis contributes roughly 50% to rate limitation, with a
``precatalytic step'' determining the remainder. The question
to be addressed now is: which of the steps in
Fig. SII
contributes most significantly to rate limitation? As
discussed by Ray
(40) , the concept of rate limitation for
enzyme-catalyzed reactions is complex. Knowles and Albery
(41) have espoused the view that there is no evolutionary reason
for enzymes to evolve such that any single step in the catalyzed
reaction is much faster than any other. In fact, EIEK and related
kinetic methods
(28) indicate that catalysis is rate-limiting
for only a few enzymes.
R transition. Converting ATCase to forms that are more R-like (either
in quaternary structure or kinetically or thermodynamically) does not
enhance the maximal rate. Examples of these include the wild-type
catalytic subunit, as well as the Tyr-240
Phe and Glu-239
Gln holoenzymes
(11, 13, 34) , which are R-like
on the basis of decreased S
(Asp) but for which
V
is not significantly different from wild-type
holoenzyme, after correcting for substrate inhibition. In addition, it
is significant that ligand-free enzyme and enzyme with ATP and CTP
bound all exhibit the same initial value of
(V/K)
at low Asp, as well as
identical hyperbolic Asp saturation curves for
(V/K)
. This indicates that all three operate
via the same enzyme form
(17) , which argues that step 1 and the
T
R transition are relatively rapid, compared to step 2. This
leaves step 2 in Fig. SIIas the prime candidate for rate
limitation.
R equilibrium
toward the T-state, does not alter V
. In
contrast, ATP does cause an increase in V
, which
is particularly evident by EIEK methods
(20) and after
correction for substrate inhibition effects. These differences suggest
that CTP alters a step in Fig. SIIthat does not contribute to
rate limitation, whereas ATP alters a step that does. Furthermore,
these differences indicate that at a detailed structural level, ATP and
CTP alter the properties of ATCase by independent mechanisms
(20) , even though at a macroscopic level they simply perturb
the T
R equilibrium in opposite directions.
Table: Summary of kinetic properties of wild-type
and Asp-236 Ala enzymes
Table: Kinetic properties of activated forms of
ATCase, compared to the Asp-236 Ala mutant, determined by
equilibrium isotope exchange kinetics upon variation of different
combinations of reactant-product pairs in constant ratio, pH 7.0, 30
°C
r
, aspartate
transcarbamoylase holoenzyme; c
, catalytic subunit of
aspartate transcarbamoylase; CP, carbamyl phosphate; Asp,
L-aspartate; CAsp, N-carbamyl-L-aspartate;
PALA, N-phosphonacetyl-L-aspartate; PIPES,
piperazine- N, N`-bis-[2-ethanesulfonic
acid]; EIEK, equilibrium isotope exchange kinetics; KIE, kinetic
isotope effects.
Ala enzyme.
The wild-type amino acid and location within the catalytic chain is
indicated to the left of the arrow, while the new amino acid is
indicated to the right of the arrow.
Ala enzyme binds Asp with weak cooperativity. Lineweaver-Burk and
Eadie-Hofstee plots were concave, and Hill plots gave n
= 1.4-1.5. Nonlinear fitting of the data according
to an equation similar to that of Pastra-Landis et al. (42) to
correct for substrate inhibition effects indicated
V
28-32 mmol/h/mg.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.