M2: Regulation of Glycogen Metabolism L7

Question 1

What are the two types of covalent modification of a protein?

Answer 1

1. Reversible

2. Irreversible

Question 2

What is a reversible covalent modification?

Give an example.

Answer 2

When a covalent bond can be undone.

Ex: Phosphorylating an enzyme using a kinase and de-phosphorylating using a phosphatase.

Question 3

What is an irreversible covalent modification?

Give an example.

Answer 3

When a covalent bond is permanently destroyed.

Ex: Pro-insulin turning into insulin via Protease. Cannot be undone.

Question 4

What is the goal of a monocyclic enzyme cascade?

Answer 4

Covalent modification of the target enzyme (makes more or less active).

Question 5

What is the goal of a bicyclic enzyme cascade? What is the advantage?

Answer 5

Goal: covalent modification of one of the modifying enzymes (which modifies target enzyme) in addition to the target enzyme.
Advantage: This allows you to fine tune signal transduction cascades bc you can regulate at multiple different points.

Question 6

What are the two regulatory mechanisms of glycogen metabolism?

Answer 6

1. Allosteric control of glycogen phosphorylase and glycogen synthase
2. Covalent modification by cascade phosphorylation (interconversion of 2 forms of the enzymes with different properties).

Question 7

Memorize the regulation of glycogen metabolism diagram.

Answer 7

L7 Slide 25.

Question 8

What activates protein kinase A (PKA)?

Answer 8

cAMP

Question 9

What does activated PKA do?

Answer 9

1. When PKA is active, it phosphorylates phosphorylase kinase b which activates it to phosphorylase kinase a. Phosphorylase kinase a activates glycogen phosphorylase b by phopshorylating it to a. This causes the breakdown of glycogen (indirect activation from PKA). It also phosphorylates glycogen synthase-a to glycogen synthase-b which causes it to be inactive and stop synthesizing glycogen (direct inhibition from PKA). This shows that phosphorylation both activates and inactivates enzymes.
2. Phosphorylates phosphoprotein phosphatase inhibitor-1 b to phosphoprotein phosphatase inhibitor-1 a (active) which sequesters phopshoprotein phsosphatase-1.

Question 10

Give an example for how a hormone, like insulin, can regulate enzyme activity in the glycogen metabolism pathway.

Answer 10

If insulin is released it can activate Phosphoprotein phosphatase-1. This would cause the dephosphorylation of glycogen synthase b (activate) which would stimulate glycogen synthesis. It would also simulate the dephosphorylation of glycogen phosphorylase a (deactivate) and of phosphorylase kinase a (deactivate) which would stop glycogen breakdown.

Question 11

What are the 2 ways that glycogen phosphorylase is regulated?

Answer 11

1. Covalent control from hormones

2. Allosteric control

Question 12

Memorize regulation of glycogen phosphorylase.

Answer 12

L7 S11.

Question 13

What state is glycogen phosphorylase b and glycogen phosphorylase a in under physiological conditions?

Answer 13

• Glycogen phosphorylase b in T state under physiological conditions (most inactive).
• Glycogen phosphorylase a in R state under physiological conditions (most active).

Question 14

Describe how the glycogen phosphorylase pathway would be effected in the fed state. What happens if you start exercising?

Answer 14

1. In the fed state, there’s a lot of ATP and/or G6P so glycogen phosphorylase-b will be mainly in inactive form (Tb).
2. In the fed state you are mostly in the Tb form. Then, if you start running, hormonal regulation (covalent control) will most probably occur before AMP muscle levels rise. Therefore, Tb will get converted to Ta and then Ra before Tb gets converted to Rb.
   (As soon as it gets converted to the Ta state it gets converted to Ra bc there aren’t many allosteric regulators in the phosphorylated form).

Question 15

What is an advantage of having glycogen phosphorylase in 4 different states in the cell at the same time? (Ra, Rb, Tb, Ta)

Answer 15

It allows for fine tuning.

Question 16

What determines the proportion of active glycogen phosphorylase in the cell under physiological conditions?

Answer 16

Under most physiological conditions, the proportion of active glycogen phosphorylase is determined by the rates of covalent modifications.

Question 17

What does phosphorylase kinase regulate when active?

Answer 17

• Activates glycogen phosphorylase by phosphorylation

- Inactivates glycogen synthase by phosphorylation

Question 18

What are the domains of phosphorylase kinase and what is bound to them when it is completely activated?

Answer 18

Domains: 4x (alpha, beta, delta, gamma)
Activated: alpha and beta are both bound by phosphate. Delta is bound by calcium. Gamma is the kinase domain (open).

Question 19

What modulates phosphorylase kinase?

Answer 19

1. Hormonal (epinephrine) via cAMP which activates PKA which activates phosphorylase kinase b by phosphorylating alpha and beta subunits.
2. Neural through release of calcium for muscle contraction and glycogen degradation. Calcium binds delta (calmodulin) subunit on phosphorylase kinase b to activate.

Question 20

When is phosphorylase kinase a in its most active state?

Answer 20

When the alpha and beta subunits are phosphorylated AND when the delta subunit is bound by calcium.

Question 21

What is the role of each domain of phosphorylase kinase?

Answer 21

• α and β: regulatory subunits are phosphorylated by PKA; dephosphorylated by PP1
• γ: catalytic subunit phosphorylates both Glycogen phosphorylase and Glycogen synthase and PP1c.
• δ: calmodulin (CaM) confers calcium sensitivity (Calcium–modulating protein)

Question 22

Describe the structure of PKA.

Answer 22

Heterotetramer with 2 catalytic subunits and 2 regulatory subunits.

Question 23

How does cAMP act on PKA? Describe it.

Answer 23

Allosterically. 2 cAMPs bind each regulatory subunit (4 cAMP total), causing the two Catalytic subunits to dissociate and act as kinases on target proteins.

Question 24

What are the target proteins of PKA?

Answer 24

Phosphorylase kinase
PP1 inhibitor
Glycogen synthase

Question 25

What are the types of glycogen synthase regulation?

Answer 25

1. Covalently

2. Allosterically

Question 26

How does covalent modification regulate glycogen synthase?

Answer 26

Phosphorylated = less active
Phosphorylated (i.e. inactivated) when:
- Phosphorylase kinase is active
- cAMP-stimulated PKA is active
- Glycogen synthase kinase (GSK3b) is active
- PP1c is inactive
  Ex: in the presence of epinephrine

Non-phosphorylated = active
Dephosphorylated (i.e. activated) when:
- PP1c is active
- Phosphorylase kinase b (inactive)
- low [cAMP]
- Glycogen synthase kinase (GSK3b) is inactive
   Ex: in the presence of insulin

Question 27

How does allosteric modification regulate glycogen synthase?

Answer 27

Only on glycogen synthase b (a is not affected).
G6P facilitates dephosphorylation of synthase b which activates it.
Ex: When G6P levels are high you are in the fed state.

Question 28

What happens to Phosphoprotein phosphatase 1 (PP1) in situations of 1. High cAMP 2. Low cAMP?

Answer 28

1. High cAMP: PKA is active and phosphorylates PP1 inhibitor-1 which inhibits PP1 by sequestering it. So, PP1 can no longer activate glycogen synthesis and deactivate glycogen breakdown.
2. Low cAMP: PKA is less active so PP1 is able to dephosphorylate its own inhibitor (PP1 inhibitor-1a).

Question 29

How does phosphoprotein phosphatase 1 catalytic subunit (PP1c) inhibit glycogen breakdown?

Answer 29

• Dephosphorylating glycogen phosphorylase
• Dephosphorylating phosphorylase kinase a
• Dephosphorylating glycogen synthase to activate Glycogen Synthesis
• Dephosphorylating it’s own inhibitory peptide : PP1-inhibitor

Question 30

What are the 3 states of PP1c activity in muscle? Describe the phosphorylation.

Answer 30

State 1: Active. Gm subunit is phosphorylated
State 0: Less active. Nothing is phosphorylated
State 2: inactive. Gm subunit is phosphorylated twice so PP1c is dissociated from Gm.

Question 31

What are the 2 subunits of PP1 and what are their functions?

Answer 31

The Gm subunit and the PP1c subunit make up PP1.

1. Gm subunit: glycogen binding subunit (middle man between PP1c and glycogen).
2. PP1c: catalytic subunit. Has phosphatase activity.

Question 32

How is PP1c hormonally regulated by insulin in the muscle?

Answer 32

Insulin (released in the fed state) activates insulin stimulated protein kinase. This causes the phosphorylation of the Gm subunit in state 0 to state 1. This allows increased glycogen synthesis.

Question 33

How is PP1c hormonally regulated by epinephrine in the muscle?

Answer 33

Epinephrine causes an increase in cAMP which activates PKA. PKA phosphorylates the second site in the Gm subunit. Converts from state 0 to state 2 and/or from state 1 to state 2 (overrides insulin fed state signal). This leads to increased glycogen breakdown and dissociation of PP1c from the Gm subunit.