Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway

I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
Like motor traffic, metabolic pathways flow more efficiently when regulated by signals. CTP, the final product of a
multistep pathway, controls flux through the pathway by inhibiting the committed step catalyzed by aspartate
transcarbamoylase (ATCase).[(Left) Richard Berenholtz/The Stock Market.]
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of
Its Pathway
Aspartate transcarbamoylase catalyzes the first step in the biosynthesis of pyrimidines, bases that are components of
nucleic acids. The reaction catalyzed by this enzyme is the condensation of aspartate and carbamoyl phosphate to form Ncarbamoylaspartate and orthophosphate (Figure 10.1). ATCase catalyzes the committed step in the pathway that will
ultimately yield pyrimidine nucleotides such as cytidine triphosphate (CTP). How is this enzyme regulated to generate
precisely the amount of CTP needed by the cell?
John Gerhart and Arthur Pardee found that ATCase is inhibited by CTP, the final product of the ATCase-controlled
pathway. The rate of the reaction catalyzed by ATCase is fast in the absence of high concentrations of CTP but decreases
as the CTP concentration increases (Figure 10.2). Thus, more molecules are sent along the pathway to make new
pyrimidines until sufficient quantities of CTP have accumulated. The effect of CTP on the enzyme exemplifies the
feedback, or end-product, inhibition mentioned earlier. Despite the fact that end-product regulation makes considerable
physiological sense, the observation that ATCase is inhibited by CTP is remarkable because CTP is structurally quite
different from the substrates of the reaction (see Figure 10.1). Owing to this structural dissimilarity, CTP must bind to a
site distinct from the active site where substrate binds. Such sites are called allosteric (from the Greek allos, "other," and
stereos, "structure") or regulatory sites. CTP is an example of an allosteric inhibitor. In ATCase (but not all
allosterically regulated enzymes), the catalytic sites and the regulatory sites are on separate polypeptide chains.
10.1.1. ACTase Consists of Separable Catalytic and Regulatory Subunits
What is the evidence that ATCase has distinct regulatory and catalytic sites? ATCase can be literally separated into
regulatory and catalytic subunits by treatment with a mercurial compound such as p-hydroxymercuribenzoate, which
reacts with sulfhydryl groups (Figure 10.3). The results of ultracentrifugation studies carried out by Gerhart and Howard
Schachman showed that p-hydroxymercuribenzoate dissociates ATCase into two kinds of subunits (Figure 10.4). The
sedimentation coefficient of the native enzyme is 11.6S, whereas those of the dissociated subunits are 2.8S and 5.8S,
indicating subunits of different size. The subunits can be readily separated by ion-exchange chromatography because
they differ markedly in charge (Section 4.1.3) or by centrifugation in a sucrose density gradient because they differ in
size (Section 4.1.6). Furthermore, the attached p-mercuribenzoate groups can be removed from the separated subunits by
adding an excess of mercaptoethanol. The isolated subunits provide materials that can be used to investigate and
characterize the individual subunits and their interactions with one another.
The larger subunit is called the catalytic (or c) subunit. This subunit displays catalytic activity, but it is not affected by
CTP. The isolated smaller subunit can bind CTP, but has no catalytic activity. Hence, that subunit is called the
regulatory (or r) subunit. The catalytic subunit, which consists of three chains (34 kd each), is referred to as c3. The
regulatory subunit, which consists of two chains (17 kd each), is referred to as r2. The catalytic and regulatory subunits
combine rapidly when they are mixed. The resulting complex has the same structure, c6r6, as the native enzyme: two
catalytic trimers and three regulatory dimers.
Furthermore, the reconstituted enzyme has the same allosteric properties as the native enzyme. Thus, ATCase is
composed of discrete catalytic and regulatory subunits, which interact in the native enzyme to produce its allosteric
behavior.
10.1.2. Allosteric Interactions in ATCase Are Mediated by Large Changes in
Quaternary Structure
How can the binding of CTP to a regulatory subunit influence reactions at the active site of a catalytic subunit?
Significant clues have been provided by the determination of the three-dimensional structure of ATCase in various forms
by x-ray crystallography in the laboratory of William Lipscomb. The structure of the enzyme without any ligands bound
to it confirms the overall structure of the enzyme. Two catalytic trimers are stacked one on top of the other, linked by
three dimers of the regulatory chains (Figure 10.5). There are significant contacts between the two catalytic trimers: each
r chain within a regulatory dimer interacts with a c chain within a catalytic trimer through a structural domain stabilized
by a zinc ion bound to four cysteine residues. The ability of p-hydroxymercuribenzoate to dissociate the catalytic and
regulatory subunits is related to the ability of mercury to bind strongly to the cysteine residues, displacing the zinc and
destabilizing this domain.
To understand the mechanism of allosteric regulation, it is crucial to locate each active site and each regulatory site in the
three-dimensional structure. To locate the active sites, the enzyme was crystallized in the presence of N(phosphonacetyl)-l-aspartate (PALA), a bisubstrate analog (an analog of the two substrates) that resembles an
intermediate along the pathway of catalysis (Figure 10.6). PALA is a potent competitive inhibitor of ATCase; it binds to
and blocks the active sites. The structure of the ATCase-PALA complex reveals that PALA binds at sites lying at the
boundaries between pairs of c chains within a catalytic trimer (Figure 10.7). Note that, though most of the residues
belong to one subunit, several key residues belong to a neighboring subunit. Thus, because the active sites are at the
subunit interface, each catalytic trimer contributes three active sites to the complete enzyme. Suitable amino acid
residues are available in the active sites for recognizing all features of the bisubstrate analog, including the phosphate
and both carboxylate groups.
Further examination of the ATCase-PALA complex reveals a remarkable change in quaternary structure on binding of
PALA. The two catalytic trimers move 12 Å farther apart and rotate approximately 10 degrees about their common
threefold axis of symmetry. Moreover, the regulatory dimers rotate approximately 15 degrees to accommodate this
motion (Figure 10.8). The enzyme literally expands on PALA binding. In essence, ATCase has two distinct quaternary
forms: one that predominates in the absence of substrate or substrate analogs and another that predominates when
substrates or analogs are bound. These forms will be referred to as the T (for tense) state and the R (for relaxed) state,
respectively. The T state has lower affinity for substrates and, hence, lower catalytic activity than does the R state. In the
presence of any fixed concentration of aspartate and carbamoyl phosphate, the enzyme exists in equilibrium between the
T and the R forms. The position of the equilibrium depends on the number of active sites that are occupied by substrate.
Having located the active sites and seen that PALA binding results in substantial structural changes in the entire ATCase
molecule, we now turn our attention to the effects of CTP. Where on the regulatory subunit does CTP bind?
Determination of the structure of ATCase in the presence of CTP reveals a binding site for this nucleotide in each
regulatory chain in a domain that does not interact with the catalytic subunit (Figure 10.9). The question naturally arises
as to how CTP can inhibit the catalytic activity of the enzyme when it does not interact with the catalytic chain. Each
active site is more than 50 Å from the nearest CTP binding site. The CTP-bound form is in the T quaternary state in the
absence of bound substrate.
The quaternary structural changes observed on substrate-analog binding suggest a mechanism for the allosteric
regulation of ATCase by CTP (Figure 10.10). The binding of the inhibitor CTP shifts the equilibrium toward the T state,
decreasing the net enzyme activity and reducing the rate of N-carbamoylaspartate generation. This mechanism for
allosteric regulation is referred to as the concerted mechanism because the change in the enzyme is "all or none"; the
entire enzyme is converted from T into R, affecting all of the catalytic sites equally. The concerted mechanism stands in
contrast with the sequential mechanism, which will be discussed shortly.
10.1.3. Allosterically Regulated Enzymes Do Not Follow Michaelis-Menten Kinetics
Allosteric enzymes are distinguished by their response to substrate concentration in addition to their susceptibility to
regulation by other molecules. Examining the rate of product formation as a function of substrate concentration can be a
source of further insights into the mechanism of regulation of ATCase (Figure 10.11). The curve differs from that
expected for an enzyme that follows Michaelis-Menten kinetics. The observed curve is referred to as sigmoid because it
resembles an "S." How can we explain this kinetic behavior in light of the structural observations? In the absence of
substrate, the enzyme exists almost entirely in the T state. However, the binding of substrate molecules to the enzyme
shifts the enzyme toward the R state. A transition from T to R favored by substrate binding to one site will increase the
enzymatic activity of the remaining five sites, leading to an overall increase in enzyme activity. This important property
is called cooperativity because the subunits cooperate with one another. If one subunit switches conformation, they all
do. The sigmoid curve can be pictured as a composite of two Michaelis-Menten curves, one corresponding to the T state
and the other to the R state. An increase in substrate concentration favors a transition from the T-state curve to the Rstate curve (Figure 10.12).
The importance of the changes in quaternary structure in determining the sigmoidal curve is illustrated nicely by studies
of the isolated catalytic trimer, freed by p-hydroxymercuribenzoate treatment. The catalytic subunit shows MichaelisMenten kinetics with kinetic parameters that are indistinguishable from those deduced for the R state. Thus, the term
tense is apt: in the T state, the regulatory dimers hold the two catalytic trimers sufficiently close to one another that key
loops on their surfaces collide and interfere with conformational adjustments necessary for high-affinity substrate
binding and catalysis.
10.1.4. Allosteric Regulators Modulate the T-to-R Equilibrium
What is the effect of CTP on the kinetic profile of ATCase? CTP increases the initial phase of the sigmoidal curve
(Figure 10.13). As noted earlier, CTP inhibits the activity of ATCase. In the presence of CTP, the enzyme becomes less
responsive to the cooperative effects facilitated by substrate binding; more substrate is required to attain a given reaction
rate. Interestingly, ATP, too, is an allosteric effector of ATCase. However, the effect of ATP is to increase the reaction
rate at a given aspartate concentration (Figure 10.14). At high concentrations of ATP, the kinetic profile shows a
lesspronounced sigmoidal behavior. Note that such sigmoidal behavior has an additional consequence: in the
concentration range where the T-to-R transition is taking place, the curve depends quite steeply on the substrate
concentration. The effects of substrates on allosteric enzymes are referred to as homotropic effects (from the Greek
homós, "same"). In contrast, the effects of nonsubstrate molecules on allosteric enzymes (such as those of CTP and ATP
on ATCase) are referred to as heterotropic effects (from the Greek héteros, "different").
The increase in ATCase activity in response to increased ATP concentration has two potential physiological
explanations. First, high ATP concentration signals a high concentration of purine nucleotides in the cell; the increase in
ATCase activity will tend to balance the purine and pyrimidine pools. Second, a high concentration of ATP indicates that
there is significant energy stored in the cell to promote mRNA synthesis and DNA replication.
10.1.5. The Concerted Model Can Be Formulated in Quantitative Terms
Conceptual Insights, Cooperative Binding and Kinetics. Interactive
graphing activities allow you to experiment with changes in the parameters and
conditions of the MWC model in order to increase your understanding of the
model and its implications for cooperative binding and kinetics.
The concerted model was first proposed by Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux; hence, it is
often referred to as the MWC model. This model can be formulated in quantitative terms. Consider an enzyme with n
identical active sites. Suppose that the enzyme exists in equilibrium between a T form with a low affinity for its substrate
and an R form with a high affinity for the substrate. We can define L as the equilibrium constant between the R and the T
forms; c as the ratio of the affinities of the two forms for the substrate, S, measured as dissociation constants; and α as
the ratio of substrate concentration to the dissociation constant K R.
Define
The fraction of active sites bound to substrate (fractional saturation, Y S) is given by
where n is the number of sites in the enzyme.
This quantitative model can be used to examine the data from ATCase, for which n = 6. Excellent agreement with
experimental data is obtained with L 200 and c 0.1. Thus, in the absence of bound substrate, the equilibrium favors
the T form by a factor of 200 (i.e., only 1 in 200 molecules is in the R form), and the affinity of the R form for substrate
is approximately 10 times as high as that of the T form. As substrate binds to each active site, the equilibrium shifts
toward the R form. For example, with these parameters, when half the active sites (three of six) are occupied by
substrate, the equilibrium has shifted so that the ratio of T to R is now 1 to 5; that is, nearly all the molecules are in the R
form.
The effects of CTP and ATP can be modeled simply by changing the value of L. For the CTP-saturated form, the value
of L increases to 1250. Thus, it takes more substrate to shift the equilibrium appreciably to the R form. For the ATP
saturated form, the value of L decreases to 70 (Figure 10.15).
10.1.6. Sequential Models Also Can Account for Allosteric Effects
In the concerted model, an allosteric enzyme can exist in one of only two states, T and R; no intermediate states are
allowed. An alternative, first proposed by Daniel Koshland, posits that sequential changes in structure take place within
an oligomeric enzyme as active sites are occupied. The binding of substrate to one site influences the substrate affinity of
neighboring active sites without necessarily inducing a transition encompassing the entire enzyme (Figure 10.16). An
important feature of sequential in contrast with concerted models is that the former can account for negative
cooperativity, in which the binding of substrate to one active site decreases the affinity of other sites for substrate. The
results of studies of a number of allosteric proteins suggest that most behave according to some combination of the
sequential and cooperative models.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.1. ATCase Reaction. Aspartate transcarbamoylase catalyzes the committed step, the condensation of
aspartate and carbamoyl phosphate to form N-carbamoylaspartate, in pyrimidine synthesis.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.2. CTP Inhibits ATCase. Cytidine triphosphate, an end product of the pyrimidine synthesis pathway, inhibits
aspartate transcarbamoylase despite having little structural similarity to reactants or products.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.3. Modification of Cysteine Residues. p-Hydroxymercuribenzoate reacts with crucial cysteine residues in
aspartate transcarbamoylase.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.4. Ultracentrifugation Studies of ATCase. Sedimentation velocity patterns of (A) native ATCase and (B)
the enzyme after treatment with p-hydroxymercuribenzoate show that the enzyme can be dissociated into regulatory and
catalytic subunits. [After J. C. Gerhart and H. K. Schachman. Biochemistry 4(1965):1054.]
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.5. Structure of ATCase. (A) The quaternary structure of aspartate transcarbamoylase as viewed from the top.
The schematic drawing at the right is a simplified representation of the relationships between subunits. A single
trimer [catalytic (c) chains, shown in orange and yellow] is visible; in this view, the second trimer is hidden
behind the one visible. (B) A side view of the complex.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.6. PALA, a Bisubstrate Analog. (Top) Nucleophilic attack by the amino group of aspartate on the carbonyl
carbon atom of carbamoyl phosphate generates an intermediate on the pathway to the formation of Ncarbamoylaspartate. (Bottom) N-(Phosphonacetyl)-l-aspartate (PALA) is an analog of the reaction intermediate and a
potent competitive inhibitor of aspartate transcarbamoylase.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.7. The Active Site of ATCase. Some of the crucial active-site residues are shown binding to the inhibitor
PALA. The active site is composed mainly of residues from one subunit, but an adjacent subunit also contributes
important residues (boxed in green).
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.8. The T-to-R State Transition in ATCase. Aspartate transcarbamoylase exists in two conformations: a
compact, relatively inactive form called the tense (T) state and an expanded form called the relaxed (R) state. PALA
binding stabilizes the R state.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.9. CTP Stabilizes the T State. The binding of CTP to the regulatory subunit of aspartate transcarbamoylase
stabilizes the T state.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.10. The R State and the T State Are in Equilibrium. Even in the absence of any substrate or regulators,
aspartate transcarbamoylase exists in an equilibrium between the R and the T states. Under these conditions, the T state
is favored by a factor of approximately 200.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.11. ATCase Displays Sigmoidal Kinetics. A plot of product formation as a function of substrate
concentration produces a sigmoidal curve because the binding of substrate to one active site favors the conversion of the
entire enzyme into the R state, increasing the activity at the other active sites. Thus, the active sites show cooperativity.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.12. Basis for the Sigmoidal Curve. The generation of the sigmoidal curve by the property of cooperativity
can be understood by imagining an allosteric enzyme as a mixture of two Michaelis-Menten enzymes, one with a high
value of K m that corresponds to the T state and another with a low value of K m that corresponds to the R state. As the
concentration of substrate is increased, the equilibrium shifts from the T state to the R state, which results in a steep rise
in activity with respect to substrate concentration.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.13. Effect of CTP on ATCase Kinetics. Cytidine triphosphate (CTP) stabilizes the T state of aspartate
transcarbamoylase, making it more difficult for substrate binding to convert the enzyme into the R state. As a result, the
curve is shifted to the right, as shown in red.
I. The Molecular Design of Life
10. Regulatory Strategies: Enzymes and Hemoglobin
10.1. Aspartate Transcarbamoylase Is Allosterically Inhibited by the End Product of Its Pathway
Figure 10.14. Effect of ATP on ATCase Kinetics. ATP is an allosteric activator of aspartate transcarbamoylase
because it stabilizes the R state, making it easier for substrate to bind. As a result, the curve is shifted to the left, as
shown in blue.