Enzyme regulation Flashcards
What are the two regulation pathways?
Enzyme abundance and substrate availability
What are the two mechanisms of controlling enzyme abundance?
- Inducible vs constituive enzymes
- Increase in rate of gene transcription
What are the two mechanisms by which substrate availability is controlled?
- 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
What is product inhibition?
Final product in a pathway inhibits an early (usually the first) step which is usually present in metabolic pathways
What is allosteric inhibition?
- 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
Does allosteric binding obey MM kinetics?
No, therefore cannot use Km but instead called [S]0.5/K0.5
What effect to allosteric activators and inhibitors have on enzyme kinetics?
activators: - Km +Vmax
inhibitors: +Km -Vmax
What is the function of asparate transcarbomylase (ATCase)?
- Catalyses step in early pyramidine biosynthesis
- carbamoyl phosphate and aspartate combine to form N-carbamoyl aspartate
How is ATCase regulated?
- 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
Name 7 methods of covalent enzyme modification
- Phosphorylation
- Covalent adenylation
- Methylation
- ADP-ribolysation
- Uridylytion
- Meristoylation
- Ubiquitination
What is the general effect of a covalent modification?
Alters local properties causing a difference in activity
What is the effect of meristoylation?
Hydrophobic myrisoyl group triggers association with the membrane
What is the effect of polyubiquitination?
Flags for proteolytic destruction
What effects can phosphorylation have on an enzyme?
- 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
What is the structure and function of enzyme glycogen phosphorylase?
- 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
How does allosteric binding affect glycogen phosphorylase?
- 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
How does AMP act as an allosteric effector of glycogen phosphorylase?
- 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
Describe the T and R state of glycogen phosphorylase
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
How does phosphorylation affect glycogen phosphorylase?
- 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
