Structural Biochemistry/Enzyme Regulation/Allosteric Control
If a cell is to make use of an enzyme, its chemical activity must be tightly monitored so that a great deal of energy will be conserved rather than wasted. Thus, like a switch, there must be a way to turn the enzyme on or off depending on when a reaction is needed. One method for regulation is allosteric control.
Allosteric control refers to a type of enzyme regulation involving the binding of a non-substrate molecule, known as the allosteric effector, at locations on the enzyme other than the active site. The name "allo" means other and "steric" refers to a position in a certain amount of space. In other words, allosteric means "at another place." An allosteric site is a site at which a small regulatory molecule interacts with an enzyme to inhibit or activate that specific enzyme; which is different from the active site where catalytic activity occurs. The binding of the allosteric effector is in general noncovalent and reversible. This interaction thus changes the shape of the enzyme which, in turn, changes the shape of the active site. This change in conformation will either inhibit or enhance the catalysis of a reaction. So the allosteric control allows the cell to regulate the needed substances quickly through inhibition and or enhancement.
The enzyme aspartate transcarbamoylase (ATCase) is an allosteric enzyme that catalyzes the first step in the synthesis of pyrimidines.
ATCase is made of six regulatory subunits and six catalytic subunits. The 3 regulatory subunits (r) are dimers made of 2 chains of 17 kd each. The smaller of the two, the regulatory subunit can bind to CTP and thus shows no catalytic activity. The 2 catalytic subunits (c) are trimers consisting of 3 chains, each 34 kd. The catalytic subunit is unresponsive to CTP thus does not follow a sigmoidal behavior.
- The quaternary structure of ATCase is composed of the two catalytic trimers stacked one on top of another. The inhibitory effect of CTP, the stimulatory action of ATP, and the cooperative binding of substates are all accompanied by large changes in the quaternary structure of ATCase.
- Each r chain of each the regulatory subunit binds with a c chain of the catalytic trimer. The region of contact on between the r chain and c chain is stabilized by a domain of zinc bound to histidine residues in the r chain. All c chains have contact with the regulatory subunit.
The catalytic and regulatory subunits can be separated by first adding a mercurial compounds and then by ultracentrifugation. Mercurial compounds breaks the connection because it displaces the Zinc ion, destabilizing the r-subunit domain. The reaction does not follow a Michaelis-Menten behavior, instead it produces a sigmoidal curve because of the response changes in the substrate concentration via regulation by other molecules and of the changes in binding probability. The addition of more substrate has two effects on increasing the probability that the enzyme will bind more than one substrate molecule while increasing the average amount of substrates bound to each enzyme. More substrate ultimately favors the R-state of ATCase since equilibrium depends on the number of active sites that are occupied by the substrate which is completely opposite of Michaelis-Menten behavior.
Allosteric enzymes exhibit sigmoidal kinetics rather than Michaelis-Menton kinetics. This is because the enzyme oscillates between two distinct conformational states, much like hemoglobin.
- The T state is characterized by low substrate affinity and low catalytic activity.
- In the R state conformation, there is a 12Å separation between catalytic trimers, and a roughly 10° rotation about central axis. There’s also a roughtly 15° rotation by the regulatory subunits. The R state conformation is characterized by an increase in substrate concentration simultaneously increasing the reactivity of ATCase in preparation for the enzymatic pathway of producing CTP.
Upon substrate binding at the active site located at pocket between the c chains in the trimer, ATCase has a greater likelihood of shifting to the R state due to substrate binding stabilizing the R state. The binding of substrates shifts the equilibrium more towards the R-state by increases the probability
that each enzyme will bind and increasing the average number of substrates bound (cooperativity).
ATP can also bind to the regulatory site of ATCase, but ATP does not inhibit the activity of ATCase in fact ATP increases the activity of ATCase. So at high levels of ATP, the ATP can act as a competitor,for the regulatory site, against CTP. So the activity of ATCase can increase with increased concentration of ATP. This increase in activity can have potential physiological explanations. High levels of ATP means that there is a high concentration of purine nucleotides, so the increased ATCase activity will increase the concentration of pyrimidines. So the concentration of both purines and pyrimidines will be more balanced. Also having a lot of ATP in the cell means having energy for processes such as mRNA synthesis and also DNA replication, so the ATCase can increase amounts of pyrimidines that can then be used in these processes.
In the presence of N-(phosphonacetyl)-L-asparate (PALA), a bisubstrate analog which resembles the substrate intermediate upon the enzymatic pathway, PALA inhibits ATCase which binds to the active sites. The inhibition however, revealed the change in the quaternary stucture upon the binding of PALA. Two catalyic trimers are isolated into their respective T and R states. This inhibition is not allosteric, but instead, introduces the catalytic subunits that are responsible for the allosteric inhibition of this complete feedback inhibition pathway.
T-state vs. R-state The T-state is known to tense up the molecule which raises the amount of substrate needed to bind to the enzyme at 1/2 Vmax (Km). T-state is less active and is favored by CTP binding. The effect of CTP is T-state becomes stabilized. This means it is harder to convert enzyme to the R-state. On the other hand, the R-state is known to be more relaxed and decreases the Km. As the concentration of the substrate increase, the equilibrium will shift from T-state to R-state. At R-state, the molecule is more active, which means substrate binding is favored. The effect of ATP on the R-state is that it is stabilized, which makes binding substrates easier.
Homotropic effects --- substrate effects on allosteric enzymes.
Heterotropic effects ---The effects of nonsubstrate molecules on allosteric enzymes such as CTP and ATP on ATCase
Cytidine triphosphate (CTP), the end product of ATCase acts as a allosteric regulator. Carbamoyl phosphate and aspartate condense into N-carbamoylaspartate intermediate which then forms CTP. CTP binds to the r chain of the regulatory subunit not in contact with the c chains. The binding of CTP stabilizes the T state and decreases substrate affinity. Even though the binding site at the regulatory subunit is distant from the catalytic subunit, the binding will result in quaternary structural changes which promotes the stabilization of the T-state and inhibition. Thus, it causes the sigmoidal curve to shift to the right. The reaction will occur fast at a low concentration of [CTP], but at higher concentrations, CTP will act as an inhibitor to ATCase by regulatory or allosteric sites, not active sites. This is an example of negative feedback, where the end result will terminate the starting reaction. The feedback inhibition of CTP on ATCase can be reversed by ATP.
Heterotropic effects --– effects of non-substrate molecules on enzyme
The rate of the product formation N-carbamoylaspartate increase as the concentration of Aspartate increases. Since it has cooperative character, you can see its curve has the sigmodal feature that means the binding of substrate on one site of the molecules increases the affinity for the other substrates to bind to the other binding sites of the molecules. The sigmodial curve of ATCase incorporates a mix of two Michaelis-Menten curves-one with a high value of KM (shown through the T state), the other with a low value of KM (shown through the R state). The binding of a substrate to a subunit and the consequent alteration of all other subunits is called cooperativity. In cooperativity, binding at one site increases or decreases binding in another site of the enzyme. This is due to the conformational changes of the neighboring sub-unit residues that affect the change in shape of the other catalytic sub-unit. This process is analogous to how hemoglobin cooperatively binds oxygen molecules.
The enzyme has two active sites. One is for substrate and the other is for the allosteric activator which is at the regulatory site. The active site of the enzyme can't bind the substrate when allosteric activators are not bound to regulatory site. On the other hand, if the allosteric activator binds to the enzyme, the shape of the active site, it enables the substrate to bind allowing products to be produced. The enzyme will remain active until the allosteric activator leaves the enzyme.
Biochemistry 6th edition. Berg, Jeremy M; Tymoczko, John L; Stryer, Lubert. W.H. Freeman Company, New York