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Proteins and Enzymes- Emily > Control of Enzyme Activity > Flashcards

Control of Enzyme Activity Flashcards

1
Q

How can enzyme activity be controlled?

A

changing the amount of enzymes, product inhibition, feedback inhibition, ligand-induced conformational change, using isozymes, by covalent modification, specific inhibitor molecules, by catalysis by cofactors, and by amplification.

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2
Q

Give an example of product inhibition.

A

Acid phosphatase by phosphate.

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3
Q

Give an example of feedback inhibition.

A

CTP and aspartate transcarbamoylase.

Hexokinase by glc-6-P.

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4
Q

Give an example of an isozyme.

A

lactate dehydrogenase.

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5
Q

Give 3 examples of covalent modification.

A

Activation of zymogens, reversible phosphorylation, and activation by thioredoxin.

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6
Q

What is allostery?

A

Binding of one ligand to enzyme/protein is affected by the binding of another (effector or modulator) at another binding site.

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7
Q

What is the homotrophic effect?

A

The ligand is identical to the substrate.

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8
Q

What is the heterotrophic effect?

A

The ligand is different to the substrate.

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9
Q

What are the different binding affinities for the substrate in the two enzyme states?

A

In the tense state (T), there is weak substrate binding. In the relaxed state (R), there is strong substrate binding.

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10
Q

What is feedback inhibition?

A

A heterotrophic inhibitory effect- the final product inhibits the enzyme.

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11
Q

What is PALA?

A

A competitive inhibitor of ATCase, which is chemically more stable than the intermediate usually formed in the reaction. It binds the R state strongly.

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12
Q

What subunits is ATCase comprised of?

A

Three regulatory dimers and two catalytic trimers.

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13
Q

How does CTP binding affect ATCase?

A

Causes heterotrophic allosteric inhibition, by stabilising the T state.

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14
Q

How can the subunits of ATCase be separated?

A

By ultracentrifugation.

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15
Q

What is the role of the zinc domain in ATCase?

A

Holds the regulatory and catalytic domains together.

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16
Q

How does ATP binding affect ATCase?

A

Causes heterotrophic allosteric positive regulation, stabilising the R state.

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17
Q

What does the MWC state?

A

Enzyme must be oligomeric. R/T forms are in equilibrium. T state is dominant in absence of the ligand. T state has a lower affinity for the ligand. Oligomer binding sites are either ALL IN T OR ALL IN R. The binding constants are also always the same.

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18
Q

What is the allosteric constant in the MWC model?

A

L = [T]/[R]

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19
Q

What is C in the MWC model?

A

The ratio of Kt and Kr

If c is small, there is a more rapid change between the T and R states.

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20
Q

What is N in the MWC model?

A

The number of subunits in the enzyme.

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21
Q

Why can the MWC model be considered to be concerted?

A

Because when one binding site changes from the T state to the R state, all binding sites change. Therefore the enzyme can only exist in two states, and the conversion between occurs in one step.

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22
Q

What are the limitations of the MWC model?

A

Suggests no immediate conformational change upon substrate binding. The binding of substrate to one site should also affect the adjacent binding sites, as a result of the conformational change. NEED THE SEQUENTIAL MODEL.

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23
Q

What does the sequential model state?

A

Enzyme must be oligomeric. Conformational changes occur sequentially at each binding site following substrate binding at the site. There are different dissociation constants for each binding ligand.

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24
Q

What is the effect of positive cooperativity in the sequential model?

A

Increase in the binding affinity for the substrate.

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25
Q

What is the effect of negative cooperativity in the sequential model?

A

Decrease in the binding affinity for the substrate.

26
Q

What are the limitations of the sequential model?

A

Suggests that the R state is only reached when all binding sites are filled and that there are 3 intermediate states between T and R. However, in experiments, when 3 of the binding sites were filled, all sites were in the R state. NEED MWC

27
Q

Describe the structure and role of myoglobin.

A

Monomeric protein with one oxygen binding site. Acts as an oxygen carrier and store in tissues.

28
Q

Describe the structure and role of haemoglobin.

A

Tetramer, with 2 α subunits and 2 β subunits. Has 4 oxygen binding sites. Acts as an oxygen carrier in blood.

29
Q

Why are oxygen carriers needed in the body?

A

Although oxygen is soluble in water, there is a limit to its solubility.

30
Q

What is heme doming?

A

The heme group is bend without oxygen binding. The group is more flat once oxygen has bound as the iron uses its d orbitals to form ionic interactions with the oxygen.

31
Q

How does heme doming explain the allostery of haemoglobin?

A

It is the driving force for the structural change in the protein, from T to R state.

32
Q

What is the hill coefficient used for?

A

To measure cooperativity.

33
Q

What are the rules of the hill coefficient?

A

n=1 no cooperativity

n is more than 1- positive cooperativity. n is less than 1- negative cooperativity.

34
Q

What is the hill plot?

A

log(θ / 1 -θ ) vs log[L] where θ is the fraction of binding sites occupied.

35
Q

What is the hill plot for myoglobin?

A

A linear plot as there is only one oxygen binding site.

36
Q

What happens to haemoglobin at low [L]?

A

Haemoglobin is in the T state and behaves as though is only has one binding site.

37
Q

What happens to haemoglobin at high [L]?

A

Haemoglobin is in the R state. However, as the ligand competes for the final binding site in each enzyme, it behaves as though it only has one binding site.

38
Q

What causes the T to R transition in haemoglobin?

A
  1. The iron (II) atom moves into the plane of the haem upon oxygen binding.
  2. The α helix containing the proximal His moves.
  3. Movement of the helix alters the interface between the αβ pairs.
  4. αβ pairs slide and rotate, forming the R state.
39
Q

How does the binding of 2,3-Bisphosphoglycerate affect haemoglobin?

A

It is a negative heterotrophic allosteric regulator. BPG favours the T state.

40
Q

How does 2,3-BPG affect fetal Hb?

A

Fetal haemoglobin has a lower affinity for BPG so binds oxygen more tightly, allowing the transfer of oxygen from maternal Hb to fetal Hb.

41
Q

What can increased 2,3-BPG levels allow?

A

Acclimitisation to high altitudes.

42
Q

What is the Bohr effect?

A

The promotion of oxygen release from haemoglobin due to decreasing pH as a result of increasing carbon dioxide levels.

43
Q

How does the Bohr effect occur?

A
  1. Carbon dioxide dissolves in plasma and crosses membranes into RBCs, where carbonic anhydrase converts carbon dioxide and water to hydrogencarbonate ions.
  2. Protonation of His by the lowered pH promotes the stabilisation of the T state in deoxyhaemoglobin.
  3. Carbon dioxide reacts with N-terminal amino groups to form carbamates, which are involved in the salt bridge that stabilises the T state.
44
Q

What are sickle cell anaemia and thalessemia?

A

Inherited autosomal recessive blood disorders, which are caused by mutations in the Hb gene and abnormal formation of haemoglobin.

45
Q

What happens to haemoglobin in sickle cell anaemia?

A

It is locked in the T state and has a low oxygen affinity.

46
Q

What are isozymes?

A

Enzymes with different primary structures and kinetic properties that catalyse identical reactions. They have different chemical equilibriums.

47
Q

What do isozymes allow?

A

Enzyme regulation in specific tissues and at various stages of development.

48
Q

What type of lactate dehydrogenase is predominately found in the heart?

A

H4, type 1.

49
Q

What type of lactate dehydrogenase is predominately found in the muscle/liver?

A

M4, type 5.

50
Q

How is H4 regulated and what reaction does it favour?

A

Feedback inhibition by pyruvate (allosteric). Favoured to oxidise lactate to pyruvate as heart muscle does not function anaroebically.

51
Q

What does M4 favour?

A

Conversion of pyruvate to lactate, allowing glycolysis to proceed anaroebically.

52
Q

What does H4 found in the blood indicate?

A

Indicates damage to heart muscle and is diagnostic of myocardial infarction.

53
Q

Which amino acids can be phosphorylated in covalent modification?

A

Serine, Threonine and Tyrosine.

54
Q

How is glycogen phosphorylase regulated?

A

By allosteric interactions and reversible phosphorylation.

55
Q

Describe the structure of glycogen phosphorylase.

A
  • 97 kDa

- dimeric with 2 binding sites

56
Q

Which form of glycogen phosphorylase is usually active and which is usually inactive?

A

Phosphorylase a is usually active (usually in R state). Phosphorylase b is usually inactive (usually in T state).

57
Q

What are the allosteric inhibitors of glycogen phosphorylase?

A

ATP, G-6-P, Glc

58
Q

What is an allosteric activator of glycogen phosphorylase?

A

AMP

59
Q

What enzymes catalyse the reversible phosphorylation of glycogen phosphorylase?

A
  • phosphorylation of b to a by phosphorylase kinase

- removal of phosphate (a to b) by phosphorylase phosphatase

60
Q

What is redox control of enzymes? Give an example of when it occurs.

A

Involves thiol/disulfide exchange.
Occurs in the Calvin cycle, as Ferredoxin-thioredoxin reductase catalyses the conversion of a thiol in the enzyme into an disulfide bride.

Proteins and Enzymes- Emily (14 decks)

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