Enzyme Regulation (Module 1 Lecture 7)

Question 0

Describe feedback inhibition.

Answer 0

1. The end product of an enzyme-catalyzed reaction inhibits the first step that begins the reaction.
2. As the product concentration changes the enzyme activity changes.
3. Considered “negative regulation.”

Question 1

What is a zymogen?

Answer 1

An inactive substance that can change conformation and become an activated enzyme (also called proenzymes)

Question 2

How are zymogens activated for digestion?

Answer 2

1. The pancreas secretes zymogens.
2. The duodenum (first part of the small intestine) secretes enteropeptidase.
3. Enteropeptidase activates trypsin.
4. Trypsin then activates other zymogens like chymotrypsin.

Question 3

Describe how chymotrypsin is proteolytically activated for digestion.

Answer 3

1. In the small intestine, trypsin cleaves one peptide bond of chymotrypsinogen (the chymotrypsin precursor), but both parts of chymotrypsin are still held together by disulfide linkages.
2. The pi-chymotrypsin part then self-cleaves into the alpha form and the other part cleaves into the b and c chains, still interconnected with disulfide bonds.

Question 4

How can you regulate the activity of an entire enzymatic pathway? Explain.

Answer 4

Because many enzymes are used in various steps of a single pathway, regulation of the entire pathway can be achieved simply by regulating the activity of one of the enzymes. For example, inhibiting enzyme 1 in the glycolysis pathway to oxidize sugar will shutdown the entire pathway.

Question 5

What are three physiological ways to regulate enzyme activity?

Answer 5

1. Allosteric control (using non-substrate molecules as modulators, like in noncompetitive inhibition), which is often used in feedback inhibition.
2. Reversible covalent modification (the most common is phosphorylation and dephosphorylation, which is used in hormonal regulation of metabolism).
3. Proteolytic activation of zymogens (digestive enzymes aren’t activated in cells but instead in the gut, and the same principal goes for blood-clotting cascade enzymes following bolog vessel injury).

Question 6

How does Allosteric regulation work and what are two examples of Allosteric proteins?

Answer 6

1. Allosteric proteins work cooperatively - they have regulatory sites distinct from their active sites.
2. When a small molecule (like signaling molecule cAMP) binds to the regulatory site it triggers a conformational change that affects the active site.
3. Examples of Allosteric proteins include ATCase (aspartate transcarbamylase) and hemoglobin.

Question 7

What is ATCase and what does it to?

Answer 7

ATCase is aspartate transcarbamylase and catalyzes the first step in pyrimidines for nucleic acid synthesis (thymine and cytosine are pyrimidines with only one ring).

Question 8

How does ATCase inhibition work?

Answer 8

1. ATCase is inhibited allosterically via feedback inhibition.
2. Cytidine triphosphate (CTP) is a final product of the pathway that winds up binding to one of ATCase’s 3 regulatory dimers.
3. The 3 regulatory dimers stop the activity of ATCase’s 2 catalytic trimers.

Question 9

Describe the kinetics of Allosteric enzymes.

Answer 9

1. Allosteric enzymes use cooperation so they don’t obey Michaelis-Menton kinetics and instead have a sigmoidal curve.
2. The curve has a slow beginning and then the slop increases as substrate binding to one site increases the activity of the other subunits on the enzyme.
3. When one subunit is isolated it will continue to obey normal kinetics.

Question 10

What are the two conformations of ATCase and what enhances each state?

Answer 10

1. The “T” state (tense and compacted) which is enhanced by the binding of CTP, the inhibitor.
2. The “R” state (relaxed and expanded) which is enhanced by the binding of substrate.

Question 11

What is cooperativity?

Answer 11

The binding of a substrate (ligand) to one subunit which will then affect the shape (and therefore the activity) of the other subunits.

Question 12

Explain the cooperativity of hemoglobin.

Answer 12

1. At the lungs the partial pressure of oxygen is around 100 torr, but it is only about 20 torr inside tissues.
2. Deoxyhemoglobin will bind to 2,3-biphosphoglycerate (2,3-BPG) to stabilize the T state and reduce hemoglobin’s affinity to oxygen.
3. This helps hemoglobin efficiently release oxygen at target tissues.

Question 13

Compare and contrast fetal and adult hemoglobin.

Answer 13

1. Fetal hemoglobin is alpha2gamma2 not alpha2beta2 tetramers like in adults.
2. Gamma subunits have reduced affinity for 2,3-BPG.
3. This difference allows oxygen to transfer from maternal to fetal red blood cells during pregnancy.

Question 14

What are three examples of reversible covalent modification?

Answer 14

1. Phosphorylation/dephosphorylation (most common).
2. Acetylation of histones (which bind to DNA to create nucleosomes.
3. Addition of long lipid chains (like myristoylation, which is irreversible and farnesylation) which anchor otherwise soluble proteins to membranes.

Question 15

Summarize how protein kinases work.

Answer 15

They transfer phosphates from ATP to the alcohol group of either serine, tyrosine, or threonine.

Question 16

How many protein kinases are there in humans and why?

Answer 16

There are over 500 human protein kinases and this gives the human body an amazing ability of protein specificity.

Question 17

What are protein phosphatases and what do they do?

Answer 17

1. They are enzymes that remove phosphates (PO4 3-) as inorganic phosphate, aka orthophosphate (Pi) from phosphorylated proteins.
2. They compete with protein kinases to determine whether or not a protein will be phosphorylated.

Question 18

Why does phosphorylation affect protein structure?

Answer 18

1. It adds to the negative charge and changes electrostatic interactions.
2. It increases the ability to hydrogen bond.
3. It increases the energy and decreases the stability of the protein which can alter the equilibrium between its tertiary and quaternary states.

Question 19

Does phosphorylation activate or deactivate enzymes? Give examples.

Answer 19

Both.

1. Some enzymes, like glycogen synthase (which catalyzes storage of sugar residues) is inactivated by phosphorylation.
2. Other enzymes, like glycogen phosphorylase (which releases stored sugar residues so that they can be metabolized) are activated by phosphorylation.

Question 20

Describe the kinase-phosphorylase cycle.

Answer 20

1. Side chain with serine, tyrosine, or threonine is phosphorylated by ATP which becomes ADP (kinase-catalyzed).
2. Phosphorylated side chain is removed as inorganic phosphate (Pi) by phosphatase and returns to it’s original serine, tyrosine, or threonine structure with alcohol.
3. Repeat step 1.

Question 21

What enzyme regulates protein kinase activity and how is it activated?

Answer 21

1. PKA (protein kinase A) is activated by cyclic AMP, which is a small signaling molecule formed by the cyclization of ATP (cAMP serves as an intercellular second messenger).
2. PKA is activated as a response to hormones epinephrine and glucagon.
3. PKA, when activated, phosphorylated many intracellular enzymes and contributes to coordinated regulation of metabolic activity.

Question 22

Describe the structure and activation mechanism of PKA.

Answer 22

1. PKA has R and C subunits (regulatory and catalytic, respectively). In it’s inactive state, the R subunits are blocking the active sites on the C subunits.
2. Four cAMP molecules bind to the R subunits.
3. The R subunits then release themselves from the catalytic subunits’ active sites, which will be free to bind and phosphorylate substrate proteins.

Question 23

Why is proteolytic activation of enzymes sometimes necessary? Give examples.

Answer 23

1. Although some enzymes acquire full activity simply by folding into the correct configurations, this is not true of zymogens.
2. Zymogens are made as inactive precursors to enzymes and can be activated later by cleavage of specific peptide bonds.
3. Two examples of this are digestive enzymes, which are dangerous if not activated inside the gut, and some peptide hormones such as insulin.
4. Proteolytic activation of enzymes is irreversible.