This course is part of a series taught by Kevin Ahern at Oregon State University on General Biochemistry. For more information about online courses go to http://ecampus.oregonstate.edu/
1. Aspartate transcarbamoylase (ATCase) is an enzyme that catalyzes the first step in pyrimidine biosynthesis (aspartate + carbamoyl phosphate N-carbamoylaspartate). This enzyme is allosterically regulated in both a positive and negative fashion and also responds to the binding of the substrate (aspartate) to it.
2. CTP, the end product of pyrimidine biosynthesis, inhibits the enzyme,whereas ATP (a purine and an indicator of high energy) activates the enzyme. This phenomenon - where the end product of a metabolic pathway inhibits the first enzyme in the pathway - is known as feedback inhibition. Feedback inhibtion is mediated allosterically - when a small molecule binds to a protein and affects the protein's activity.
3. ATCase has 12 subunits - 6 catalytic and 6 regulatory. The smaller regulatory subunits bind CTP, but not the catalytic subunits.
4. Binding of CTP to the regulatory subunits of ATCase causes the enzyme to stabilize (lock) in the T state (tight, less reactive state). In the T state, the quaternary structure of the enzyme the enzyme exhibits reduced affinity for substrate. In the opposite state, the R state (relaxed state, more reactive state), the enzyme has increased activity and a higher affinity for substrate. ATP stabilizes the R state of the enzyme.
5. In the absence of ATP and CTP, the enzyme can freely flip between the T and R states, but the T state tends to predominate.
6. If one removes all of the ATP and CTP from the enzyme and starts adding increasing amounts of the enzyme's normal substrate (aspartate) and then measures the reaction rate (velocity), one discovers a sort of sigmoidal plot, which indicates that the enzyme is actually changing as more aspartate is added to it. The actual change is a flip from the T state to the R state stabilized by the aspartate.
7. You should be familiar with the effects of ATP, CTP and increasing aspartate on ATCase.
8. A manmade artificial substrate, known as PALA, binds to ATCase and inhibits the enzyme, but interestingly locks it in the R state. Though the enzyme can't catalyze a reaction when bound to PALA, it is clear that its form changes, again indicating that enzymes are changed by the binding of substrate and that T and R states exist.
9. Protein kinase A is an enzyme involved in covalent modification of enzymes. Like all kinases, it catalyzes the addition of a phosphate to a molecule. Since it is a protein kinase, the molecules it attaches a phosphate to are protein molecules.
10. Phosphates get attached to hydroxyl side chains in proteins. Protein kinase A attaches phosphate to serine or threonine. Other protein kinases can attach phosphate to tyrosine side chains.
11. Protein kinase A is controlled by allosteric means. It is composed of two regulatory subunits and two catalytic subunits. The overall complex is called R2C2. When the catalytic subunits are bound to the regulatory subunits, they cannot catalyze reactions. However, when the molecule cAMP binds to the regulatory subunits, the catalytic subunits are released and are thus active. When active, they covalently modify proteins by adding phosphates.
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