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Protein Control in Cellular Regulation > Mechanisms of Protein Regulation > Flashcards

Mechanisms of Protein Regulation Flashcards

1
Q

How can enzyme activity be regulated?

A
  1. Changes in substrate concentration
  2. Binding of small effector molecules
  3. Reversible covalent modifications
  4. Binding of regulatory proteins
  5. Proteolytic activation
  6. Controlling the amount of enzyme present
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2
Q

How does changing the substrate concentration affect enzyme activity?

A

It affects the rate of the enzyme-catalyzed reaction. An increase in substrate concentration leads to an increase in the rate.

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3
Q

When does the substrate concentration affect enzyme activity?

A

When substrate concentration is less than Km, the rate is linearly dependent on substrate concentration.

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4
Q

What are isoenzymes?

A

Enzymes that catalyze the same reaction but have a different amino acid sequence.

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5
Q

How are isoenzymes made?

A

Encoded by different genes or derived by alternative splicing from one gene.

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6
Q

Are isoenzymes the same?

A

Yes and no as they can have different kinetic/regulatory properties.

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7
Q

How do the different forms of lactate dehydrogenase compare?

A

Each contain four polypeptides composed of two different types of protein.

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8
Q

What are the main forms of lactate dehydrogenase? Where do they exist?

A

H4 (heart) and M4 (skeletal muscle).

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9
Q

What does lactate dehydrogenase do?

A

It catalyzes the reversible conversion of lactate to pyruvate with the reduction of NAD+ to NADH and vice versa.

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10
Q

How do the reactions of H4 and M4 compare?

A

H4 favours lactate oxidation (pyruvate is final product for energy) and M4 favours pyruvate reduction (lactate final product).

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11
Q

How do the reactions of H4 and M4 compare for Km?

A

Both low Km, but H4 for lactate and M4 for pyruvate.

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12
Q

What is allosteric regulation?

A

Regulation mediated by interactions of a modulator at a regulatory site away from the active/binding site.

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13
Q

What does the binding of an allosteric modulator do?

A

Causes a change in conformation that affects the activity of the enzyme.

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14
Q

Homotropic modulator

A

The modulator is the same as the substrate in terms of size and shape.

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15
Q

Heterotropic modulator

A

The modulator is different from the substrate.

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16
Q

How do allosterically regulated enzymes exist?

A

They are usually multi-subunit proteins.

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17
Q

How do allosterically regulated enzymes exist in Michaelis-Menten kinetics?

A

They don’t obey the kinetics.

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18
Q

What is co-operative binding?

A

The binding of the substrate to the enzyme increases the association reaction, so that each incoming substrate binds easier.

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19
Q

What happens to hemoglobin when oxygen binds to it?

A

It promotes the stabilization of the R state.

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20
Q

What are the differences between T state and R state?

A

The T state has low affinity for oxygen whereas the R state of hemoglobin has a higher affinity and usually occurs when a ligand is bound.

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21
Q

Allosteric activators

A

Increase the proportion of enzyme in the R state.

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22
Q

Allosteric inhibitors

A

Increase the proportion of enzyme in the T state.

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23
Q

When are covalent modifications made?

A

Post-translationally, after the protein is formed.

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24
Q

Are covalent modifications permament?

A

No, most are reversible.

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25
Q

Give an example of covalent modifications.

A

Phosphorylation, acetylation, ubiquitination, etc.

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26
Q

What do protein kinases do?

A

They transfer the terminal phosphate from ATP to the -OH group of ser, thr, and tyr.

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27
Q

What do protein phosphatases do?

A

Reverse the effects of kinases by catalysing the hydrolytic removal of phosphoryl groups from proteins.

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28
Q

What can the addition of a phosphoryl group do?

A

It can form three or more hydrogen bonds that allow for specific interactions with new hydrogen bond donors.

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29
Q

What does the negative charge of phosphate do?

A

It disrupts the existing electrostatic interactions and may allow new ones to form of the enzyme.

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30
Q

What does phosphorylation do in terms of energy?

A

It provides a great amount of energy to allow a shift in the confirmation of the phosphorylated protein.

31
Q

What does phosphorylation do for cell signalling?

A

It allows amplification cascades.

32
Q

How do the kinetics of phosphorylation work?

A

They can be adjusted from a few seconds to several hours.

33
Q

What does phosphorylation do to the structure?

A

It affects the molecule through allosteric activation.

34
Q

Explain the effects of protein phosphorylation on glycogen phosphorylase.

A
  1. The phosphorylation of glycogen phosphorylase results in a high activity form (R state)
  2. The dephosphorylation of glycogen phosphorylase (2 phosphates removed) results in a low activity form (T state)
35
Q

What are common sequences for protein kinases?

A

Serine and threonine.

36
Q

What does amplification of kinase cascades do?

A

Allows amplification of the initial signal by several orders of magnitude within a few milliseconds. It occurs when enzymes activate enzymes, thus the number of affected molecules increase.

37
Q

Explain amplification of a kinase cascade in the liver.

A

Adrenergic signalling in the liver regulates the glycogen metabolism through a cascade of enzymes:
1. 1 epinephrine molecule binds to a receptor complex
2. This activates 10 G s alpha subunits
3. Increase of adenylyl cyclase, which causes the increase in 200 cAMP
4. Activation of 100 PKA
5. Activation of 1000 phosphorylase b kinase
6. Activation of 10,000 phosphorylase a kinase
7. Glycogen is converted to glucose 1-phosphate (100,000 molecules)
8. Over 100,000 glucose molecules transported into the blood

38
Q

How are protein kinases activated?

A

The binding of small molecules and proteins.

39
Q

Explain the activation of PKA.

A
  1. Epinephrine binds to a beta-adrenergic receptor
  2. This activates the GPCR
  3. G s alpha subunit replaces GDP with GTP and dissociates
  4. The activated G s alpha subunit activates adenylyl cyclase
    –> many Gs alpha subunits may be activated by one occupied receptor
  5. Adenylyl cyclase turns ATP to cAMP
  6. cAMP activates PKA
40
Q

How is the activation of PKA reversed?

A

When cAMP is degraded by cyclic nucleotide phosphodiesterase.

41
Q

How is the activity of PKA blocked in the absence of cAMP?

A

A pseudosubstrate region which binds the R subunit to the catalytic site blocks activity (R2C2).

42
Q

How does the pseudosubstrate sequence differ from the PKA consensus sequence?

A

An alanine instead of serine.

43
Q

How does cAMP activate PKA?

A
  1. 2 molecules of cAMP bind to each R subunit (4 cAMP in total)
  2. Change in R subunit confirmation causes the release of C subunits
  3. Free C subunits car catalytically active
44
Q

What are AKAPs?

A

A-Kinase anchoring proteins that have different domains specific proteins that allow for signals to be spatially restricted in the cell.
ex. AKAP5 holds PKA, PP2A (phosphatase), the target protein, the GPCR, and adenylyl cyclase.

45
Q

Explain the activation of the calcium release pathway.

A
  1. Hormone binds to a GPCR
  2. Activated GTP-G q alpha subunit dissociates
  3. It binds to PLC to cleave PIP2 in the plasma membrane, resulting in IP3 and DAG
  4. IP3 binds to a specific receptor-gated calcium channel in the ER, releasing calcium into the cytoplasm
  5. DAG and the release of calcium activate PKC at the surface of the plasma membrane
  6. Phosphorylation of cellular proteins by PKC produces some of the cellular responses to the hormone
46
Q

How are proteins regulated by calcium?

A
  1. 4 calcium bind to calmodulin
  2. The complex changes conformation to become activated
  3. Then, the two globular “hands” of the complex wrap around a binding site on a target protein (adenylyl cyclase, MLCK, NO synthase, etc,)
47
Q

What does calcium bind to in calmodulin?

A

Calcium binds between 2 alpha helices and a short loop, binding to 7 oxygen atoms.

48
Q

What is a zymogen?

A

Longer inactive form of an enzyme.

49
Q

What is regulation by partial proteolysis?

A

The removal of a N terminal pro-segment by breaking a peptide bond releases the fully active form.

50
Q

Why is partial proteolysis a highly specific process?

A

Breaking of a peptide bond in the zymogen is catalyzed by an enzyme.

51
Q

What isn’t involved in partial proteolysis?

A

ATP

52
Q

Can partial proteolysis be reversed?

A

Nope, it’s irreversible

53
Q

Give examples of proteins that are activated by partial proteolysis.

A

Activation of trypsin (trypsinogen is inactive) using enteropeptidase to remove a segment. Also, activation of chymotrypsin.

54
Q

How are activated zymogens switched off?

A

Tight binding of inhibitors and degradation.

55
Q

How to change the rate of protein synthesis?

A

Enzyme induction/repression.

56
Q

How to change the rate of protein degradation?

A

Ubiquitin-proteasome pathway.

57
Q

Explain the multiple modes of regulation that allow control of phosphofructokinase-1.

A
  1. Allosteric inhibitors inhibit
  2. Allosteric activators activate
  3. Hormonal regulation inactivate/activate
  4. Transcriptional control inactivate/activate
58
Q

How are metabolic pathways controlled?

A
  1. Controlling the amount of enzymes
  2. Controlling the availability of substrates
  3. Controlling the catalytic activity of enzymes through feedback regulation
59
Q

Negative feedback regulation

A

A product in the pathway is used to reduce an enzyme activity earlier on.

60
Q

Positive feedback regulation

A

A product in the pathway is used to increase an enzyme’s activity earlier on.

61
Q

What kinetics do allosteric enzymes/modulators display?

A

Sigmoidal kinetics

62
Q

What kinetics does Michaelis-Menten display?

A

Hyperbolic kinetics

63
Q

What happens if the substrate has limited availbility?

A

The total cellular concentration of the substrate is constant, but the ratio of the two coenzymes can vary. Therefore, one can be changed into the either when more is needed. Ex. NAD+ can be changed to NADH and vice versa when needed

64
Q

What happens to the electrostatic interactions when moving for T-state to R-state?

A

In T-state, histidine usually interacts with aspartate/glutamate. However, when in R-state, hemoglobin is rotated 30 degrees, breaking this interaction and shifting histidine to the middle.

65
Q

What happens to the curve when an allosteric activator is added?

A

Shifts to the left, which means the enzyme is more active at lower substrate concentrations.

66
Q

What happens to the curve when an allosteric inhibitor is added?

A

Shifts to the right, which means the enzyme is less active at lower substrate concentrations.

67
Q

What states does hemoglobin exist in when deoxygenated?

A

In both the T and R-state; however, more T-state hemoglobins’ exist.

68
Q

What covalent modification isn’t made post-translationally?

A

The carboxylation of thrombin. As it’s being synthesized in the ER, the carboxyl group is added, thus non-reversible too.

69
Q

What is the committed step?

A

The first irreversible, unique step in a metabolic pathway under physiological conditions; this step is catalyzed by an allosteric enzyme and commits the product to a particular chemical fate. Also known as the rate-limiting step.

70
Q

What does CTP do?

A

It acts as an allosteric feedback inhibitor of ATCase.

71
Q

Why is CTP a feedback inhibitor?

A

Since it’s the product of the metabolic pathway, it controls the committed step (ATCase). It ensures that intermediates aren’t needlessly formed when pyrimidines are abundant.

72
Q

How does ATP behave in the ATCase metabolic pathway?

A

It’s an allosteric activator as it binds to the same site as CTP (different from the active site), and increases the reaction rate, shifting the enzyme to its R-state.

73
Q

What molecular interactions are involved in allosteric regulation?

A

Non-covalent ones between the regulatory molecule and the site on the enzyme.

Protein Control in Cellular Regulation (8 decks)

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