Enzyme regulation

Question 1

What are the two regulation pathways?

Answer 1

Enzyme abundance and substrate availability

Question 2

What are the two mechanisms of controlling enzyme abundance?

Answer 2

• Inducible vs constituive enzymes

- Increase in rate of gene transcription

Question 3

What are the two mechanisms by which substrate availability is controlled?

Answer 3

• Non-regulatory enzymes show hyperbolic kinetics (MM) where at low substrate concentration, reaction rate proportional to substrate concentration
• Regulatory enzymes show sigmoidal kinetics where changes of substrate concentrations at normal physiological levels greatly alter reaction rate

Question 4

What is product inhibition?

Answer 4

Final product in a pathway inhibits an early (usually the first) step which is usually present in metabolic pathways

Question 5

What is allosteric inhibition?

Answer 5

• Regulated by reversible/non-covalent binding of regulatory allosteric modulators/effectors
• Binding of positive modulator causes conformtational change on C subunit allowing binding of S

Question 6

Does allosteric binding obey MM kinetics?

Answer 6

No, therefore cannot use Km but instead called [S]0.5/K0.5

Question 7

What effect to allosteric activators and inhibitors have on enzyme kinetics?

Answer 7

activators: - Km +Vmax
inhibitors: +Km -Vmax

Question 8

What is the function of asparate transcarbomylase (ATCase)?

Answer 8

• Catalyses step in early pyramidine biosynthesis

- carbamoyl phosphate and aspartate combine to form N-carbamoyl aspartate

Question 9

How is ATCase regulated?

Answer 9

• Regulatory subunits have binding sites for ATP (+) and CTP (-)
• CTP produced at the end of the pyramidine pathway, binds to revert complex to less active T state
• ATP indicates robust cellular metabolism, and so binds to upregulate DNA trancription/replication

Question 10

Name 7 methods of covalent enzyme modification

Answer 10

• Phosphorylation
• Covalent adenylation
• Methylation
• ADP-ribolysation
• Uridylytion
• Meristoylation
• Ubiquitination

Question 11

What is the general effect of a covalent modification?

Answer 11

Alters local properties causing a difference in activity

Question 12

What is the effect of meristoylation?

Answer 12

Hydrophobic myrisoyl group triggers association with the membrane

Question 13

What is the effect of polyubiquitination?

Answer 13

Flags for proteolytic destruction

Question 14

What effects can phosphorylation have on an enzyme?

Answer 14

• can cause major changes in the conformation of the modified enzyme: steric effects, hydrogen bonding and ionic interactions to neighboring residues
• This can lead to effects on substrate binding and catalysis

Question 15

What is the structure and function of enzyme glycogen phosphorylase?

Answer 15

• Catalyses the phosphorylation of glycogen to glucose-1-P which is either metabolised in the muscle or converted to glucose in the liver
• Dimer of two identical subunits where each subunit has an active sitewhich contains a pyridoxal cofactor covalently attached via a Schiff base

Question 16

How does allosteric binding affect glycogen phosphorylase?

Answer 16

• Inorganic phosphate binds cooperatively allowing large increases in activity
• glucose-1-phosphate is isomerised into glucose-6-phosphate which enters the glycolytic pathway to produce ATP which is a feedback inhibitor of glycogen phsophorylase

Question 17

How does AMP act as an allosteric effector of glycogen phosphorylase?

Answer 17

• Competes for same binding site as ATP but has positive effect on the cooperativity of substrate binding
• Higher AMP levels indicate low ATP levels and so an increase in glycolysis is needed

Question 18

Describe the T and R state of glycogen phosphorylase

Answer 18

T state - active site buried, Asp-283 faces active site causing electrostatic repulsion
R state - Exposed active site as Asp-283 is replaced with Arg-569 causing favourable electrostatic interactions

Question 19

How does phosphorylation affect glycogen phosphorylase?

Answer 19

• In crisis conditions allosteric controls are overridden by reversible phosphorylation via enzyme phsophorylase kinase
• Phosphorylation at Ser-14 converts enzyme to more active, allosterically unresponsive a state