Control of Enzyme Activity Flashcards
What is the rate limiting step?
Reactants are abundant while products are undetectable (slowest step)
With no rate limiting step, all reactions are close to equilibrium and are thermodynamically driven towards the end
How is catalytic activity controlled?
- Enzyme availability
By gene expression/protein degradation
This mechanism is slow (mins/hours)
Used to regulate ‘blocks of genes’ or whole pathways
Product inhibition - this is fast/reversible (however, a large amount of excess product is needed) - Allosteric activation/inhibition by small molecules
Affects existing enzyme population
Very fast and temporary meaning reversible (1 sec) - Allosteric activation/inhibition by phosphorylation
Mediated by protein kinases – covalent modification, serine, tyrosine and histidine
Regulated by external signals (hormones)
Fast activation (1 min)
In principle long lived as reversible by phosphatases
What is the molecular basis of allosteric regulation?
Allosteric - meaning other solid/site
This is distant form the active site, and could be within a separate regulatory subunit
Allosteric enzymes:
They are composed of multiple subunit enzymes, and therefore multiple substrate binding sites
They tend to exhibit additional allosteric binding sites for regulation/phosphorylation
Subunits cooperate with each other - binding and catalysis on different
What are some examples of allosteric mechanisms?
- Haemoglobin (binding cooperativity, regulation of O2 affinity by allosteric effectors such as BPG and protonation=pH change - the Bohr effect)
- Aspartate transcarbamoylase (allosteric inhibition)
- Glycogen phosphorylase (phosphorylation)
- Hexameric helicases (RNA-induced catalytic cooperativity)
Describe ATCase?
Aspartate Transcarbamoylase
6 catalytic subunits with 6 regulatory subunits in between (12 subunits)
3-fold symmetry - trigonal bipyramidal
2 different substrates
ATCase is the first enzyme unique to the synthetic pathway of pyrimidine phosphates (e.g. CTP, UTP)
This is a good regulatory point as it is the first step in this pathway
ATCase catalyses the reaction: Carbamoyl phosphate + Aspartate -> N-carbamoylaspartate + H2PO4-
What is the control of regulation involved in ATCase?
Allosterically inhibited by CTP (product of the biosynthetic pathway), a pyrimidine nucleotide
Negative feedback to prevent overproduction
Allosterically activated by ATP (product of the purine nucleotide biosynthetic pathway)
Positive feedback to maintain purine and pyrimidine nucleotides in balance for nucleic acid synthesis
PALA - a substrate analogue that is involved in inhibition - binds to the R-state
How is activation/inhibition of ATCase accomplished?
It is accomplished by changing the affinity for successive binding of substrate molecules
Sigmoidal kinetics/binding curve = cooperativity
Binding of the first substrate molecule is less favoured than that of the subsequent - therefore positive cooperativity, as the Hill coefficient > 1
The apparent KM is shifted in the presence of allosteric effector
What is the molecular mechanism of ATCase?
Each subunit exists in two states:
T(=tight/tense) with low substrate affinity
R(=relaxed) with high substrate affinity
Transition T to R - involves subunit rotation
Upper and lower trimer get further apart (11Å) and rotate by 12 degrees
Regulatory dimers rotate by 15 degrees and separate by 4Å
Interactions between interfaces of the subunits are altered
The T to R transition closes the substrate binding pocket
Binding of activator (ATP) to the regulatory subunit stabilizes the R state
T state is stabilized by inhibitor (CTP) binding to the allosteric site
What is phosphorlyation regulation?
Covalent modification:
The phosphorlyation/dephosphorylation of the hydroxyl group of either serine, threonine or tyrosine residues
What proteins are involved in phosphorlyation regulation?
Protein kinase - phosphorylates and leads to activation
Protein phosphatase - dephosphorylates and leads to inactivation
Phosphorylation stabilises the active (R) form
Give an example of an enzyme controlled by covalent modification?
In response to hormone signalling: stress cAMP mediates reversible phosphorylation, this leads to increased glucose availability (fight/flight response)
Glycogen phosphorylase: involved in the rapid replenishment of glucose reserves, catalysing the breakdown of glycogen into glucose-1-phosphate
It is activated by reversible phosphorylation in response to signalling pathway
The enzyme exists in two states: active: A and inactive: B
There are additional allosteric effectors e.g. AMP in order to further modulate the activity
What is the molecular mechanism of activation of glycogen phosphorlyase?
- Phosphorylation of Ser 14 - causes refolding of N-terminal tail – which in turn allows AMP binding
- AMP binding - causes favourable tower helix reorientation
- Reorientation of catalytic site residues into the active conformation T->R state
What are hexameric RNA helicases?
Important in RNA virus - to order the viral RNA into the helical packages
It runs along the RNA, to unwind the genetic material
What is the co-operativity of RNA helicase?
Co-operativity results from coordination of ATP hydrolysis within the hexamer
Both Km and Kcat are affected
There is a Hill coefficient of 2.7 - almost three or more subunits cooperate
Inhibition with a non-hydrolysable ATP analogue reveals that three neighbouring subunits cooperate in hydrolysis
The different conformations were found by using different allosteric effectors
What is an example of an allosteric enzyme?
hSOS1 - human son of sevenless 1
It is a multicomplex enzyme but not a multi-subunit enzyme
SOS is a guanine nucleotide exchange factor
Responsible for activating Ras in response to binding of a growth factor to a receptor tyrosine kinase (e.g. epidermal growth factor receptor)
Catalyses exchange of Ras.GDP (inactive) to Ras.GTP (active)
