LECTURE 7 - GLYCOGEN 2 Flashcards
what are the two types of regulation?
non covalent (allosteric)
covalent regulation
what are the two types of covalent modifications of a protein?
reversible: when a covalent bond can be undone (kinase and phosphatase)
irreversible: when a covalent bond is permanently destroyed
(pro-insulin and insulin)
understand monocyclic enzyme cascades
aim: covalent modification of the target enzyme
understand bicyclic enzyme cascades
aim: covalent modification of one of the modifying enzymes in addition to the target enzyme
what is special about kinases and phosphatases?
have multiple substrates
what is an example of a bicyclic enzyme cascade?
the target is to activate glycogen phosphorylase, which is activated by phosphorylase kinase, which first has to be activated by protein kinase A (PKA)
what are the two regulatory mechanisms of glycogen metabolism?
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)
little reminder: what does glycogen phosphorylase do?
breaks down glycogen to release G1P
what are the two conformations of glycogen phosphorylase and how are they controlled?
T state (tense): inactive
R state (relaxed): active
the conformations are controlled by allosteric effectors
in glycogen phosphorylase, what is controlled by allosteric vs what is controlled by covalent?
allosteric control is for the R-T transition
covalent control tunes the T form
what does covalent control of glycogen phosphorylase do?
goes from most inactive state to a little active state
2 phosphorylations by phosphorylase kinase
what does allosteric control of glycogen phosphorylase do, and how?
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
how does the protein go from the most inactive to the most active state?
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
what determines the proportion of glycogen phosphorylase under most physiological conditions?
determined by the rate of covalent modification (phosphorylations)
what does phosphorylase kinase do?
- activates glycogen phosphorylase for glycogen breakdown by phosphorylation
- inactivates glycogen synthase by phosphorylation
how is phosphorylase kinase organised?
16 individual proteins, organised in 4x4x4x4 subunits
what are the different subunits of phosphorylase kinase?
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
what are the two forms of phosphorylase kinase?
phosphorylase kinase beta: inactive, but active when calcium is elevated
phosphorylase kinase alpha: active even when there is low calcium
what happens when phosphorylase kinase is activated?
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
what are the two inputs through which phosphorylase kinase is modulated?
- hormonal, (epinephrine), via cAMP
- neural through release of calcium for muscle contraction and glycogen degradation
what is PKA dependent on?
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
what are the target proteins of PKA?
- phosphorylase kinase
- the inhibitor of PP1
- glycogen synthase
PKA structure and how it is activated?
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
what does glycogen synthase do?
the enzyme that builds up glycogen for UDP glucose
how does phosphorylation affect glycogen synthase?
phosphorylate=less active
non phosphorylate=active
what does glycogen synthase activity depend on?
the fraction of the enzyme in the unmodified form
when is glycogen synthase phosphorylated?
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
when is glycogen synthase dephosphorylated?
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
what are allosteric regulations of glycogen synthase?
only on glycogen synthase b (a is not affected)
G6P facilitates dephosphorylation of synthase b
when is phosphoprotein 1 (PP1) inactive?
when it is bound to its inhibitor, PP1 inhibitor a
this is allosteric, it’s a protein protein interaction
what activates the PP1 inhibitor?
PKA and PP1c
this phosphorylates and activates the inhibitor
how can PP1 reactivate?
it can reactivate itself, because not the entire proportion of the protein is bound to the inhibitor + the reaction is reversible
how does PP1c inhibit glycogen breakdown?
-dephosphorylates glycogen phosphorylase
- dephophorylates phosphorylase kinase a
- dephosphorylates glycogen synthase to activate glucogen synthesis
- dephosphorylates it’s own inhibitory peptide, PP1-inhibitor
which proteins lead to the degradation of glycogen?
PKA
phosphorylase kinase
glycogen phosphorylase
GSK3b
which proteins lead to the synthesis of glycogen?
glycogen synthesis
phosphoprotein phosphatase 1 (PP1)
PP1 inhibitor
entire map of the regulation
how is PP1c attached to glycogen?
the catalytic subunit is attached to the Gm subunit (regulatory)
the regulatory portion is bound to glycogen
where are the glycogen regulating proteins bound?
they are not free floating in the cell, they are always bound to glycogen, but are either working or not working
what happens to PP1c when you go to a fed state?
- 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
what happens to PP1c when you go to a fasting state/ you are running?
- 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
