Allosteric Enzyme Regulation
The Wonderful World of ATCase
Aspartate Transcarbamoylase (ATCase) catalyzes the first step in the biosynthesis of pyrimidine (future lecture topic). This was the first allosteric enzyme to be extensively studied.
From the class lecture slide, here’s the specific reaction:
CTP is the end product from a reaction whose first step is catalyzed by ATCase. It turns it out that CTP is an inhibitor of ATCase: as [CTP] rises, ATCase activity decreases, an example of feedback inhibition. Scenarios where the end product of a pathway regulate the early steps of the pathway, thus limiting wasteful production of that end product, is common in catabolic pathways.
That CTP inhibits ATCase is remarkable because CTP has no structural similarity to the substrate of ATCase, as would be the case with a competitive inhibitor.
Etymology: Greek, “allos” = other, “ster” = structure: enzymes that can adopt other structures.
Allosteric Enyzmes have regulator sites distinct from catalytic sties.
Because of the structural dissimilarity, allosteric sites must be different from the active sites. Allosteric regulation then refers to the binding of an allosteric effector to the regulatory subunits of the enzyme. They can be either activators or inhibitors.
Back to ATCase. By purifying ATCase in a sucrose gradient and treating with a special compound, it’s possible to isolate two peaks–meaning, the enzyme as two subunits: a regulatory unit (green) that binds the inhibitor and a catalytic subunit (blue) that binds the substrate and converts to the product.
Here’s its structure:
More specifically, ATCase is made of six catalytic subunits organized into two catalytic trimers and six regulatory subunits of the enzyme in three dimers.
Upon binding to aspartate, ATCase changes conformation from a T-state to an R-state, where:
- T-state: ATCase is inactive.
- R-state: ATCase is active, binding of substrate stabilized.
As will be shown, the binding of CTP favors the T-state, thus slowing enzyme activity.
Kinetic features of ATCase
Experiment: mix ATCase with one substrate held constant, then let the other vary. From this determine the V0 at various concentrations.
Observed: S-curve like binding of Hb to O2. Shape is “sigmoidal.”
The S-curve indicates cooperativity: when [ASP] is low, an increase in [ASP] has negligible effect until it passes a threshold, at which point the velocity rapidly increases as [ASP] increases. Then at a high [ASP], the enzyme saturates and approaches Vmax.
Back to this experiment, replace ATCase with purified catalytic subunits (no regulatory subunits) and suddenly the curve becomes hyperbolic. This is the behavior of a classic Michaelain enzyme. Thus, no cooperativity.
This is an example of homotropic allosteric behavior: the binding of the substrate stabilizes the R-state of the enzyme.
This in contrast to the behavior of CTP, which binds to the regulatory unit to inhibit the enzyme, an example of heterotropic allosteric behavior: the binding of a molecule other than the substrate allosterically regulates the enzyme. Here, it’s an inhibitor.
Allosteric enzymes do not follow the Michaelain-Menten equation. Thus, there’s a different notion of (inverse) affinity, Km.
For allosteric enzymes, it’s called Ko.s. = the [substrate] at which V0 = 1/2 Vmax. CTP, then, raises Ko.s.–the higher the value, the lower the affinity.
ATP, on the other hand, activates ATCase by stabilizing the R-state. Ko.s. decreases.
Why does ATP activate ATCase? As will be shown in later sections, a high concentration of ATP (from a physiological point of view), means that there’s a correspondingly high concentration of purine synthesis happening; thus, the shift to pyrimidine synthesis can become activated. Secondly, it means there’s enough energy in the cell to promote mRNA synthesis and DNA replication.
- CTP: inhibitor of ATCase, stabilizes its T-state, increases Ko.s.
- ATP: activate of ATCase, stabilizes its R-state, decreases Ko.s.
Monod, Wyman, Changeux (MWC) Model
assumptions: allosteric proteins are oligomeric, made of several subunits, arranged in a symmetrical fashion. Each has one binding site per ligand. The enzyme exists in at least two different conformations: RR and S state (in equilibrium).
Turns out the assumption does not always hold.
Koshland, Nemethy, Filmer (KNF) Model
assumptions: a sequential progression from T->R instead of an all or nothing event. If no ligand bound to the enzyme, the enzyme has a single conformation, T. When one binds, a change in conformation occurs limited to subunit This change of conformation is transmitted to neighboring subunits. Progressively changes to final R state.
every variation of conformation is possible. KNF and MWC are limiting cases. MWF predicts response to change in Aspartate. KNF predicts respond to CTP and ATP.