Regulation - Keeping The System In Balance

Question 1

Why does the cellular factory need to be regulated?

Answer 1

Because we need to regulate how much carbon goes towards each metabolic process. Regulate catabolism Vs anabolism

Question 2

What are the two mechanisms by which regulation is achieved?

Answer 2

Mass action

Feedback loops

Question 3

What are the mechanisms by which enzyme activity is regulated?

Answer 3

Allosteric regulation
Spatial clustering
Post translational covalent modification

Question 4

Specific example of allosteric regulation of enzyme activity

Answer 4

Inhibition of ATCase action by the CTP product

Question 5

Specific example of spatial clustering regulation

Answer 5

Biosynthesis of inosine monophosphate (IMP)

In deficiency of purines, enzymes respond by forming puranosomes which is associated with 50% increase in flux of purine formation pathway

Question 6

Specific example of post translational covalent modification regulation

Answer 6

Phosphorylation of PDC enzyme (catalyses pyruvate to acetyl CoA) by kinase produced less active form of enzyme so decreases rate of its catalysis and acetyl CoA production

Dephosphorylation of PDC enzyme by phosphatase produced more active form of enzyme so increases rate of its catalysis and acetyl CoA production

Question 7

Explain how CTP inhibits ATCase

Answer 7

CTP binds to an allosteric site on ATCase

Question 8

How are the catalytic units in ATCase arranged?

Answer 8

6 catalytic sub units arranged as a pair of trimers which sit on top of each other

Question 9

How are the regulatory units in ATCase arranged?

Answer 9

6 regulatory subunits arranged as three dimers that sit between each junction of the catalytic subunits

Question 10

Where does CTP bind on ATCase?

Answer 10

CTP binds to an allosteric site on each regulatory subunit

Question 11

On the trimer of catalytic subunits, where are the active sites located?

What does each active site contain?

Answer 11

At the junctions between the three catalytic subunits (three active sites)

Each active site contains key AA residues from each of the two catalytic subunits that it is in between

Question 12

What state is the enzyme ATCase in when CTP is bound?

Activity?

Answer 12

T (tight) state

Less active

Question 13

What state is the enzyme ATCase in when CTP is NOT bound?

Answer 13

R (relaxed) state

More active

Question 14

When CTP is NOT bound and ATCase is in a relaxed state … rotation thing

Answer 14

Catalytic subunits are rotated 10 degrees about their common axis of symmetry

This brings the key AAs at the junctions of the subunits closer together which improves the rate of catalysis

Question 15

When CTP IS bound and ATCase is in a tight state … distance thing

Answer 15

Catalytic trimers are moved 1.2 nm apart which provides better substrate access to active sites

Question 16

What type of kinetic curve does ATCase have

Answer 16

Sigmoid all

Question 17

Explain why ATCase has a sigmoidal curve

Answer 17

It is the combination of the two states (relaxed and tight) of the enzyme

Question 18

Which enzyme regulates the feedback loop involved in regulating anabolic and catabolic reactions of glycolysis

Answer 18

Phosphofructokinase

Question 19

What does phosphofructokinase catalyse

Answer 19

Fructose-6-phosphate

To

Fructose-1,6-biphosphate

Question 20

What happens if ADP concentation is too high

Answer 20

ADP too high so activates PFK enzyme and rest of glycolysis

Overrides feedback loop so pfk noblonger inhibited, continues to makes more carbon skeleton to put into TCA cycle to increase ATP synthesis

Question 21

What happens if too much PEP

Answer 21

Negative feedback loop = PEP binds allosterically to PFK and inhibits it

Question 22

Typically, metabolic pathways contain…

Answer 22

Multiple feedback and feedforward loops

Important in metabolic engineering - trying to break this loops to make it do something it doesn’t normally do

Question 23

Kinetic regulation by mass action

Answer 23

Mathematical model predicts changes in substrate concentration

More substrate = enzyme goes faster

As concentration of glucose increase, point where there’s no longer an increase in flux because we have reached Vmax of enzymes in system.

Question 24

In mass action at which point is the kinetic response of the system limited?

Answer 24

When we reach the Vmax of the enzyme sin the system and the active sites of all the enzymes are saturated

Question 25

What happens when you saturate available enzyme capacity

Answer 25

xx don’t get

Question 26

Mass action

XX???

Answer 26

Kinetic propagation in response to variable glucose levels

Question 27

Realistic organisation of metabolic pathway in the cell

Answer 27

Diffusing all the time
Moving around
Many copies of each enzyme

Question 28

What’s a purinosome

Answer 28

Bodies of enzymes normally dispersed through the cell

Question 29

Why is there an increase is rate of catalysis and bio synthetic pathway for purine formation when purinosomes form?

Answer 29

Because when the enzymes are all clustered it means there’s a LOCAL increase in concentration. So within the complex there’s a higher substrate concentration so Vmax is higher, so reaction goes faster as each substrate moves into enzyme etc)

Question 30

Advantage of regulation through spatial organisation of enzymes

Answer 30

Enables control over branch points in pathways

Question 31

Example of spatial organisation of enzymes helping control branch points in pathways

Answer 31

Branching point = one direction goes on to synthesise purines, other direction goes on to synthesise thiamine

If starved of purine we want to go the direction that synthesises purine

So all enzymes involved in purine synthesis put into cluster. This means it’s naturally more likely for the substrate that leaves one enzyme in this pathway to go to the next enzyme involved in this pathway rather than to leave the cluster and go to another enzyme which would take it to the thiamine pathway.

Question 32

Explain feedback loop if TCA cycle is producing too much NADH

Answer 32

Too much NADH

Need to slow flow of pyruvate into cycle so need to slow catalyses of pyruvate to acetyl coA

NADH activates kinase

Kinase phosphorylates enzyme making it less active so less catalysis of pyruvate to acetyl CoA

Question 33

Explain feedback loop if pyruvate is building up

Answer 33

Tells us enzyme (PDC) is going too slow

Pyruvate inhibits kinase

Kinase no longer phosphorylase’s enzyme making it inactive

Question 34

What is phosphorylation controlled by

Answer 34

Kinases

Question 35

What is dephosphorylation controlled by

Answer 35

Phosphorylase’s

Question 36

How does phosphorylation produce amplified effects

Answer 36

One kinase can phosphorylase lots of target proteins Ina. Short amount of time

Question 37

How is phosphorylation linked to cells energy status

Answer 37

It uses ATP to get the phosphate so if cell is depleted in ATP phosphorylation won’t go ahead if it doesn’t need to

Question 38

What does adding a phosphoryl group do?

Answer 38

Adds two negative charges and can form 3 or more H bonds.

These changes alter structural conformation of the enzyme and affect substrate binding and catalytic activity

Question 39

Why can’t binding of CTP to ATCase be competitive inhibition

Answer 39

because CTP canot fit into active site of ATCase due to diff shape, size and charge