Background
What is phenylalanine?
- Phenylalanine is an essential amino acid
# 1 From Case Study: Is phenylalanine glucogenic, ketogenic, or both?
Phenylalanine is both glucogenic and ketogenic. It is glucogenic because one of the products of the degradation pathway is fumarate, which can be converted to OAA which can enter gluconeogenesis to form glucose. A second product is acetoacetate, a ketone body, so phenylalanine is also ketogenic amino acid.
#3 From Case Study: Once the enzyme was purified, the investigators set out to determine its properties. They wanted to see if phenylalanine, in addition to serving as a substrate for the enzyme, had an additional role in the regulation of the enzyme. Polyacrylamide gels (under denaturing and non-denaturing conditions) were carried out with the rat liver PAH. The results are shown in Figure 30.2. How do you interpret these data?
PAH is made of identical subunits, each about 49,000 daltons. When treated with SDS, the protein breaks apart into individual subunits, showing one band—meaning all the subunits are the same.
Under normal (non-denaturing) conditions, PAH appears as a dimer (two subunits together), since its size is about twice that of a single subunit.
When PAH is incubated with phenylalanine, it forms a tetramer (four subunits together), as shown by a band that is four times the size of one subunit.
**In short:** PAH normally exists as a dimer but becomes a tetramer in the presence of phenylalanine.
In an experiment, increasing phenylalanine concentration beyond a certain point does not further increase velocity. What does this indicate?
The enzyme is saturated and Vmax has been reached.
Where does phenylalanine come from?
- It comes from dietary proteins such as meat, dairy, eggs, etc.
#5 From Case Study:
Kinetic data in which PAH activity is compared with and without preincubation with phenylalanine is shown in Figure 30.3. Give a structural basis for the interpretation of these data.
Preincubating PAH with phenylalanine changes it from a dimer to a tetramer.
The tetramer binds the substrate better, which is why the reaction goes faster.
Dimer = T form → low affinity (binds poorly)
Tetramer = R form → high affinity (binds well)
#4 From Case Study:
Next, kinetic studies were carried out with the enzymes. A plot of velocity vs. phenylalanine concentration yields a sigmoidally shaped curve. What does this tell you about the enzyme?
PAH is normally a dimer, but in high levels of phenylalanine it becomes a tetramer.
The S-shaped (sigmoidal) curve shows cooperative binding: When one phenylalanine binds, it makes it easier for more to bind.
This means PAH is an allosteric enzyme, so its activity can be turned up or down by molecules that regulate how fast phenylalanine is broken down.
Preincubation with phenylalanine increases enzyme activity. Provide a structural explanation for this observation.
Phenylalanine stabilizes the active conformation of PAH, shifting the equilibrium toward a more active state.
What is the importance of phenylalanine?
- Our bodies use it to make proteins and other important molecules
#7 From Case Study:
Tyrosine is not an essential amino acid in normal persons, but it is essential in persons with PKU. Explain why.
Essential amino acids must come from the diet because the body can’t make them.
Nonessential amino acids can be made by the body.
Normally, the body can make tyrosine (Tyr) from phenylalanine (Phe), so tyrosine isn’t essential.
But in people with PKU, this conversion doesn’t happen (they lack the enzyme), so they can’t make tyrosine.
Because of this, tyrosine becomes essential and must be included in the diet.
Addition of phenylalanine changes the banding pattern in non-denaturing gels. What does this suggest about its role?
Phenylalanine induces a conformational change, promoting a more active form of the enzyme.
If a PAH enzyme showed a Michaelis-menten curve instead of a sigmoidal, what would this suggest about its regulation?
It would suggest the enzyme is not allosteric and does not exhibit cooperative binding.
What does the PAH enzyme normally do?
- Phenylalanine hydroxylase (PAH) converts phenylalanine into tyrosine
- This is the first step in breaking down phenylalanine
#8 From Case Study:
Patients with the disease PKU tend to have blue eyes, fair hair, and very light skin. Explain why.
Phenylalanine is converted to tyrosine in normal individuals. The tyrosine is then converted to melanin, which is the compound responsible for pigment in skin and hair.
Gel electrophoresis shows a single band under denaturing conditions but multiple bands under non-denaturing conditions. What does this indicate about PAH structure?
PAH has the same subunit but exists in multiple quaternary structures under regular conditions.
Why does glucagon, but not insulin, activate PAH? Relate your answer to metabolic states.
Glucagon is active during fasting, promoting amino acid breakdown for energy, while insulin promotes anabolic pathways, so it does not activate PAH.
What happens when PAH is missing?
Phenylalanine can't be converted into tyrosine, so
- Phenylalanine builds up, which can get converted into a toxic substance
- Can harm brain development and neurotransmitter production
Why is controlling phenylalanine levels important in people with PKU, and how is this managed?
In PKU, the body cannot properly break down phenylalanine because PAH doesn’t work correctly. This causes phenylalanine to build up, which can be harmful. To manage this, people with PKU must follow a low-phenylalanine diet and often supplement with tyrosine, since they cannot make it themselves.
#6 From Case Study:
The effects of the hormones glucagon and insulin on PAH activity were investigated. The results are shown in Figure 30.4. In addition, the amount of radioactively-labeled phosphate incorporated into PAH with glucagon treatment was found to be nearly seven-fold greater than in controls.
a. How would you interpret these data?
b. Which hormone activates PAH, and why?
Insulin decreases PAH activity, while glucagon increases it.
Glucagon likely works by turning on a protein kinase (via cAMP), which phosphorylates PAH and makes it more active. Glucagon and phenylalanine work together (synergistically), so the enzyme is most active when both are present.
When blood sugar is low, glucagon levels are high. This may activate PAH to break down phenylalanine into products (like fumarate) that can be used to make glucose (gluconeogenesis).
#2 From Case Study: In order to learn more about the PAH enzyme, it was necessary to purify it. PAH has been isolated from both rats and humans. In the rat, three isozymes of PAH have been identified in the liver. Their molecular weights are identical, but their charges differ, as isoelectric focusing demonstrates. The pI values are 5.2, 5.3 and 5.6. DEAE-cellulose (anion exchange) chromatography was one of the steps in the purification procedure of the enzymes. Predict the order of elution of these isozymes from the DEAE-cellulose column. What pH buffer would you choose in running the column?
DEAE-cellulose has positively charged beads, so it attracts negatively charged proteins.
Proteins with low pI (more acidic) are more negative → they stick more → elute last
Proteins with high pI are less negative (or positive) → they stick less → elute first
So, the proteins come off in this order: 5.6 → 5.3 → 5.2
A buffer around **pH 5.3** is used because:
* Protein with **pI < 5.3** → negative
* Protein with **pI > 5.3** → positive
* Protein with **pI = 5.3** → neutral
This helps separate them better.