aspects of metabolic regulation

Question 1

what amino acids are proteins phosphorylated on (known for decades)?

Answer 1

Ser, Thr, Tyr

on hydroxyl side chain

Question 2

what has more sensitive spectrometry revealed?

Answer 2

acetylation of lys
hydroylation of pro
o glycosylation of serine and more

Question 3

How does protein kinase work?

Answer 3

transfer phoshphoryl group from ATP to aser thr or try residue

Question 4

How does protein phosphatase work?

Answer 4

remove phosphoryl group using water to remove Pi

Question 5

How does protein kinase A work?

Answer 5

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

Question 6

Pyruvate Kinase Inhibitors

Answer 6

ATP
ACetyl coa 
long fatty acids 
alanine
Glucagon ( activates cAMP dependent protein kinase which phosphorylates pyruvates kinase inactivating it)

Question 7

Pyruvate kinase Activators?

Answer 7

Fructose 1-6 bisphosphate

Question 8

How are pathway intermediates kept?

Answer 8

Low concentrations and far from equilibrium

Question 9

What three reactions exibit large decreases in free energy.

sites of allosteric control?

Answer 9

Hexokinase
Phosphofructokinase
Pyruvates kinase

Question 10

What does PFK-1 do?

Answer 10

catalyses the commited step of glycolysis

is the tap that turns on and off glycolysis

Question 11

What inhibits PFK-1?

Answer 11

ATP

Citrate

Question 12

What activates PFK-1?

Answer 12

AMP

Fructose 2-6 biphosphate

Question 13

How does energy status affect PFK-1?

Answer 13

PFK1 high activity low energy status

Low activity high enegry status

Question 14

How does hill number work?

Answer 14

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

Question 15

Difference between R and T state?

Answer 15

R state: high activity relaxed state
T state: low activity
Tense state

Question 16

What does PFK2 catalyse?

Answer 16

formation fructose 2,6-bisphosphate

Question 17

What does FBPase catalyse?

Answer 17

dephosphorylation of Fructose 2,6 bisphophate back to Fructose 6 phosphate.

Question 18

Does PFK2 indirectly regulate PFK1?

Answer 18

Yes

Question 19

How many subunits does Human PFK1 have?

Answer 19

4 subunits

Tetramer

Question 20

What kinds of interactions are found in the PFK1 tetramer?

Answer 20

Hydrophobic and electrostatic interactions.

Question 21

what does the ATP bound state represent in PFK1?

Answer 21

R- State

Question 22

What does the ADP bound state represent in PFK1?

Answer 22

T- State

Question 23

What is the effect of Fructose 26 bisphosphate on PFK1 and FBPase acitivty?

Answer 23

Stimulates PFK1 activity

Reduces FBPase activity

Question 24

How could you describe 6 phosphofructo-2-kinase/ fructose-2,6- bisphosphatase?

Answer 24

Bifunctional enzyme structure

Question 25

What does phosphorylation of PKA at residue 29 do to6 phosphofructo-2-kinase/ fructose-2,6- bisphosphatase?

Answer 25

Activation of F2,6bPase

Inactivation of PFK2

Question 26

What does Phosphorylation by AMPK at residue 466 do to 6 phosphofructo-2-kinase/ fructose-2,6- bisphosphatase?

Answer 26

Activation of PFK2

innactivation of F2,6bPase

Question 27

What does adenylate kinase do?

Answer 27

ADP is the substrate for the enzyme adenylate kinase catalyses the reaction
2ADP ⇋ ATP + AMP

Question 28

How many subunits does AMPK have?

Answer 28

3

Question 29

what does the Alpha subunit in AMPK do?

Answer 29

catalytic subunit
Thr172 can be phosphorylated
acitvates kinase

Question 30

what does the Beta subunit in AMPK do?

Answer 30

Targets kinase to different regions in the cell

Question 31

What does the Gamma subunit in AMPK do?

Answer 31

Regulatory

Question 32

In AMPK what promotes R state?

Answer 32

AMP and ADP

AMP more

Question 33

In AMPK what promotes T state?

Answer 33

ATP

Question 34

How is AMPK phosphorylated and activated?

Answer 34

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.

Question 35

How does Calmodulin dependent kinases work?

Answer 35

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

Question 36

How odes LKB1 work

Answer 36

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

Question 37

How does overall pathway work?

Answer 37

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

Question 38

What can tissue do if its running short on oxygen?

Answer 38

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

Question 39

How can NO affect muscle relaxation

Answer 39

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.