LECTURE 7 - GLYCOGEN 2

Question 1

what are the two types of regulation?

Answer 1

non covalent (allosteric)
covalent regulation

Question 2

what are the two types of covalent modifications of a protein?

Answer 2

reversible: when a covalent bond can be undone (kinase and phosphatase)

irreversible: when a covalent bond is permanently destroyed
(pro-insulin and insulin)

Question 3

understand monocyclic enzyme cascades

Answer 3

aim: covalent modification of the target enzyme

Question 4

understand bicyclic enzyme cascades

Answer 4

aim: covalent modification of one of the modifying enzymes in addition to the target enzyme

Question 5

what is special about kinases and phosphatases?

Answer 5

have multiple substrates

Question 6

what is an example of a bicyclic enzyme cascade?

Answer 6

the target is to activate glycogen phosphorylase, which is activated by phosphorylase kinase, which first has to be activated by protein kinase A (PKA)

Question 7

what are the two regulatory mechanisms of glycogen metabolism?

Answer 7

glycogen metabolism has 2 distinct pathways, either synthesis or degradation, depending on physiological needs

regulatory mechanisms:
1. allosteric control of glycogen phosphorylase and glycogen synthase
2. covalent modification by cascade phosphorylation (interconversion of 2 forms of the enzymes which have different properties)

Question 8

little reminder: what does glycogen phosphorylase do?

Answer 8

breaks down glycogen to release G1P

Question 9

what are the two conformations of glycogen phosphorylase and how are they controlled?

Answer 9

T state (tense): inactive
R state (relaxed): active
the conformations are controlled by allosteric effectors

Question 10

in glycogen phosphorylase, what is controlled by allosteric vs what is controlled by covalent?

Answer 10

allosteric control is for the R-T transition
covalent control tunes the T form

Question 11

what does covalent control of glycogen phosphorylase do?

Answer 11

goes from most inactive state to a little active state
2 phosphorylations by phosphorylase kinase

Question 12

what does allosteric control of glycogen phosphorylase do, and how?

Answer 12

when there is high ATP and/or G6P the protein will go from R form to T (no need for glucose)
when there is high AMP, exercise, the protein will go from T to R

Question 13

how does the protein go from the most inactive to the most active state?

Answer 13

by the combination of phosphorylation AND allosteric control
it first goes to the little bit inactive state because putting on a phosphate is faster than allosteric control

Question 14

what determines the proportion of glycogen phosphorylase under most physiological conditions?

Answer 14

determined by the rate of covalent modification (phosphorylations)

Question 15

what does phosphorylase kinase do?

Answer 15

• activates glycogen phosphorylase for glycogen breakdown by phosphorylation
• inactivates glycogen synthase by phosphorylation

Question 16

how is phosphorylase kinase organised?

Answer 16

16 individual proteins, organised in 4x4x4x4 subunits

Question 17

what are the different subunits of phosphorylase kinase?

Answer 17

alpha and beta: regulatory subunits, phosphorylated by PKA, dephosphorylated by PP1
gamma: catalytic subunit, phosphorylates both glycogen phosphorylase and glycogen synthase
delta: calmodulin (present in many proteins), confers calcium sensitivity

Question 18

what are the two forms of phosphorylase kinase?

Answer 18

phosphorylase kinase beta: inactive, but active when calcium is elevated

phosphorylase kinase alpha: active even when there is low calcium

Question 19

what happens when phosphorylase kinase is activated?

Answer 19

activated by protein kinase A and calcium
all 8 proteins of the alpha and beta subunits are phosphorylated
calcium is on the delta subunit (needed for muscle contraction)

this can be fine tuned

Question 20

what are the two inputs through which phosphorylase kinase is modulated?

Answer 20

1. hormonal, (epinephrine), via cAMP
2. neural through release of calcium for muscle contraction and glycogen degradation

Question 21

what is PKA dependent on?

Answer 21

PKA is cAMP dependent
intracellular concentrations of cAMP determine the fraction of PKA in the active form
the activation of PKA does not depend on covalent modifications

Question 22

what are the target proteins of PKA?

Answer 22

• phosphorylase kinase
• the inhibitor of PP1
• glycogen synthase

Question 23

PKA structure and how it is activated?

Answer 23

PKA is a heterotetramer
two regulatory subunits and two catalytic subunits
cAMP binds the two regulatory subunits, causing the two catalytic subunits to dissociate and act as kinases on the target proteins
the regulatory subunits have pockets for cAMP binding

Question 24

what does glycogen synthase do?

Answer 24

the enzyme that builds up glycogen for UDP glucose

Question 25

how does phosphorylation affect glycogen synthase?

Answer 25

phosphorylate=less active non phosphorylate=active

Question 26

what does glycogen synthase activity depend on?

Answer 26

the fraction of the enzyme in the unmodified form

Question 27

when is glycogen synthase phosphorylated?

Answer 27

phosphorylated (inactivated) when: phosphorylase kinase is active cAMP-stimulated PKA is active glycogen synthase kinase (GSK3b) is active PP1c is inactive - in the presence of epinephrine

Question 28

when is glycogen synthase dephosphorylated?

Answer 28

dephosphorylated (activated) when PP1c is active phosphorylase kinase b is inactive low concentration of cAMP glycogen synthase kinase (GSK3b) is inactive - in the presence of insulin

Question 29

what are allosteric regulations of glycogen synthase?

Answer 29

only on glycogen synthase b (a is not affected) G6P facilitates dephosphorylation of synthase b

Question 30

when is phosphoprotein 1 (PP1) inactive?

Answer 30

when it is bound to its inhibitor, PP1 inhibitor a this is allosteric, it's a protein protein interaction

Question 31

what activates the PP1 inhibitor?

Answer 31

PKA and PP1c this phosphorylates and activates the inhibitor

Question 32

how can PP1 reactivate?

Answer 32

it can reactivate itself, because not the entire proportion of the protein is bound to the inhibitor + the reaction is reversible

Question 33

how does PP1c inhibit glycogen breakdown?

Answer 33

-dephosphorylates glycogen phosphorylase - dephophorylates phosphorylase kinase a - dephosphorylates glycogen synthase to activate glucogen synthesis - dephosphorylates it's own inhibitory peptide, PP1-inhibitor

Question 34

which proteins lead to the degradation of glycogen?

Answer 34

PKA phosphorylase kinase glycogen phosphorylase GSK3b

Question 35

which proteins lead to the synthesis of glycogen?

Answer 35

glycogen synthesis phosphoprotein phosphatase 1 (PP1) PP1 inhibitor

Question 36

entire map of the regulation

Answer 36

Question 37

how is PP1c attached to glycogen?

Answer 37

the catalytic subunit is attached to the Gm subunit (regulatory) the regulatory portion is bound to glycogen

Question 38

where are the glycogen regulating proteins bound?

Answer 38

they are not free floating in the cell, they are always bound to glycogen, but are either working or not working

Question 39

what happens to PP1c when you go to a fed state?

Answer 39

- you produce insulin - insulin will bind its protein kinase, which will result in phosphorylations - the Gm subunit will be phosphorylated, and PP1c will be active - this leads to glycogen synthesis

Question 40

what happens to PP1c when you go to a fasting state/ you are running?

Answer 40

- epinephrine will be released - an increase of cAMP will activate protein kinase A, which will result in some phosphorylations - the Gm subunit will be phosphorylated twice - the PP1c catalytic subunit dissociates, and the protein is inactive - this will lead to increased glycogen breakdown - PP1c can go from active to inactive by phosphorylating the Gm subunit one more time