aspects of metabolic regulation Flashcards
what amino acids are proteins phosphorylated on (known for decades)?
Ser, Thr, Tyr
on hydroxyl side chain
what has more sensitive spectrometry revealed?
acetylation of lys
hydroylation of pro
o glycosylation of serine and more
How does protein kinase work?
transfer phoshphoryl group from ATP to aser thr or try residue
How does protein phosphatase work?
remove phosphoryl group using water to remove Pi
How does protein kinase A work?
protein has 3 subunits Catalytic (c) regulatory(R) and anchoring(AKAP)
AKAP allows the kinase to be targeted to the vicinity of its substrates
in innactive complex R subunit R inserts inhibitor sequence into c subunit
2 cAMP molecules can bind to each R this releases C subunits no longer inhibited to kinase is activated
Pyruvate Kinase Inhibitors
ATP ACetyl coa long fatty acids alanine Glucagon ( activates cAMP dependent protein kinase which phosphorylates pyruvates kinase inactivating it)
Pyruvate kinase Activators?
Fructose 1-6 bisphosphate
How are pathway intermediates kept?
Low concentrations and far from equilibrium
What three reactions exibit large decreases in free energy.
sites of allosteric control?
Hexokinase
Phosphofructokinase
Pyruvates kinase
What does PFK-1 do?
catalyses the commited step of glycolysis
is the tap that turns on and off glycolysis
What inhibits PFK-1?
ATP
Citrate
What activates PFK-1?
AMP
Fructose 2-6 biphosphate
How does energy status affect PFK-1?
PFK1 high activity low energy status
Low activity high enegry status
How does hill number work?
Hill number close to subunits in protein= strong interactions
Hill number smaller eg 4 subunit protein with hill number 1 there would be no interaction between subunits and would therefore follow Michaelis Menten
Difference between R and T state?
R state: high activity relaxed state
T state: low activity
Tense state
What does PFK2 catalyse?
formation fructose 2,6-bisphosphate
What does FBPase catalyse?
dephosphorylation of Fructose 2,6 bisphophate back to Fructose 6 phosphate.
Does PFK2 indirectly regulate PFK1?
Yes
How many subunits does Human PFK1 have?
4 subunits
Tetramer
What kinds of interactions are found in the PFK1 tetramer?
Hydrophobic and electrostatic interactions.
what does the ATP bound state represent in PFK1?
R- State
What does the ADP bound state represent in PFK1?
T- State
What is the effect of Fructose 26 bisphosphate on PFK1 and FBPase acitivty?
Stimulates PFK1 activity
Reduces FBPase activity
How could you describe 6 phosphofructo-2-kinase/ fructose-2,6- bisphosphatase?
Bifunctional enzyme structure
What does phosphorylation of PKA at residue 29 do to6 phosphofructo-2-kinase/ fructose-2,6- bisphosphatase?
Activation of F2,6bPase
Inactivation of PFK2
What does Phosphorylation by AMPK at residue 466 do to 6 phosphofructo-2-kinase/ fructose-2,6- bisphosphatase?
Activation of PFK2
innactivation of F2,6bPase
What does adenylate kinase do?
ADP is the substrate for the enzyme adenylate kinase catalyses the reaction
2ADP ⇋ ATP + AMP
How many subunits does AMPK have?
3
what does the Alpha subunit in AMPK do?
catalytic subunit
Thr172 can be phosphorylated
acitvates kinase
what does the Beta subunit in AMPK do?
Targets kinase to different regions in the cell
What does the Gamma subunit in AMPK do?
Regulatory
In AMPK what promotes R state?
AMP and ADP
AMP more
In AMPK what promotes T state?
ATP
How is AMPK phosphorylated and activated?
One of these is activated by rise in cytoplasmic Ca2+ concentrations: Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKβ).
Another is LKB1, which phosphorylates AMPK in response to AMP binding to the AMPK γ subunit.
How does Calmodulin dependent kinases work?
memory molecules
Ca pulse activates CAMKKbeta. This phosphorylates AMPK activating AMPK. A short ca pulse can therefore give sustained activation of AMPK the ca pulse has been remembered
How odes LKB1 work
AMP bind to gamma subunit promotes AMPK phosphorylation.
AMP initiates assembly of AMPK activating complex increases affinity of AMPK fot the linker protein AXIN that binds LKB1
Complex brings LKB1 to vicinity of AMPK prpmoting phosphorylation of AMPK at Thr 172
How does overall pathway work?
Vigorous exercise leads to rapid ATP turnover. Adenylate kinase (AK) supports [ATP]. AK activity results in a rise in [AMP]
Increasing [AMP] activates AMPK.
AMPK phosphorylates and activates PFK-2 (PFKFB2, slides 33, 35)
PFK-2 catalyses formation of F-2,6-bP.
F-2,6-bP activates PFK-1, making more F-1,6-bP available for glycolysis.
Note also that AMPK probably stimulates Glut4 and increases supply of glucose (see BI520
What can tissue do if its running short on oxygen?
AMP will increase which will activate AMPK which promotes NO formation by phosphoryalting eNOS NO results in vasodilation that allows more oxygen to be delivered to for ox phos
How can NO affect muscle relaxation
No diffuse from endothelial cells through to smooth muscle.
activates guanylate cyclase stimulates production of cGMP
cGMP activates protein kinase G
inhibits contaction leading to muscle relaxation increased blood flow.
