Lecture 16 - Enzyme regulation - allosteric regulation Flashcards
How can enzymes be regulated?
Enzymes can be regulated by:
* On/Off transcriptional control, factors that should be considered are
○ Enzyme half lives
○ Rate of transcription
○ Rate of protein synthesis
Enzyme regulation is dynamic and different mechanisms of regulation will operate over different time scales. For example allosteric regulation occurs over short time scales in comparison to covalent modification (phosphorylation), compartmentalisation and aggregation which all occur over moderate time scales. The longer timescales are changes in gene expression.
What is allosteric regulation and define cooperativity.
Allosteric regulation - when a small molecule (or ligand) binds to an enzyme inducing a conformational change in the shape of the enzyme and its active site. The change in shape effects the activity of the enzyme
* The conformational change is powerful, rapid and reversible
* Conformational change provides a basis for regulating gene expression as well as enzyme activity
* Allosteric regulators bind to a separate regulatory site which can either have a positive or negative effect on regulation by either making the active site available or unavailable to the substrate
* Multiple different types of regulatory sites are possible
* Substrates and regulators can be very different in structure
Allosteric proteins exhibit Cooperativity - activity at one functional site affects activity at other functional sites
If an enzyme has multiple subunits with multiple active sites and regulatory sites binding by a modulator at one can cause a change in shape in multiple of these subunits
Allosteric regulation by a positive regulator
1. The enzyme is less active and the substrate fits poorly into the active site/low affinity
2. The positive modulator binds which changes the shape of the active site increasing the affinity for the substrate increasing enzyme activity
3. The active enzyme substate complex forms when the substrate binds
Describe allosteric regulation in E.coli aspartate transcarboamylase (ATCase)
The allosteric enzyme that catalyses on of the first committed steps of pyrimidine biosynthesis
Carbamoyl phosphate + aspartate —-> N-carbamoylaspartate —-(additional steps)—-> CTP (Pyrimidine)
* The ATCase is positively regulated by ATP (Purine)
* It is negatively regulated by CTP through feedback inhibition
Structure of E. Coli ATCase
It is composed of two catalytic trimers stacked on top of each other surrounded by 3 regulatory dimers.
The regulatory domain contains a zinc domain which is essential for correct folding
Experimental interrogation of ATCase structure
* P-hydroxymercuribenzoate reacts with Cys side-chains which is found in the regulatory subunit and causes a loss of zinc binding which disrupts the quaternary structure of ATCase
* Sedimentation analysis
Migrate to form two peaks one made up of R subunits and one made up of catalytic subunits
What is meant by the tensed and relaxed states of allosteric enzymes.
Allosteric enzymes exist in two states the tensed and relaxed state. In the relaxed states the catalytic units are further apart and have rotated slightly in respect to one another. In the Tensed less active form the affinity for the substrate is decreased. In the relaxed state there is a higher affinity.
Describe the mechanistic effect of allosteric activation and inhibition of ATCase.
- ATCase does not follow Michaelis-Menton kinetics it exhibits sigmoidal kinetics (s-shaped curve). MM kinetics can be seen with only the catalytic subunits but the allosterically regulated form displays sigmoidal kinetics.
- The sigmoidal curve of allosterically regulated enzymes is the sum of the R-state and T-state curves.
- Negative regulators (CTP in the case of ATCase) pulls towards the T-state curve as more substrate is needed
- Positive regulators pull the curve towards the R-form (left) - less substrate is needed.
Quantitative formulation of Monod’s concerted model and the enzymology of ATCase
* Fractional activity (Y) - Fraction of active sites bound to the substrate
* α - ratio of [S] to KR when E is in R-state
* L - ratio of E in T-state to that in the R-state
* Binding of regulators changes l and thus the response to the substrate
C - KR/KT - Difference in affinity
Describe the two models that account for allosteric behaviour
Concerted
○ Allosteric enzymes exist in only two states - all subunits are in same state, and these states (T and R) are in equilibrium
○ Upon binding a ligand the enzyme is stabilised in one of these conformation. - shifts equilibrium
Sequential
○ The binding of a ligand doesn’t necessarily cause all subunits to be in the same state however it changes the likelihood of binding a certain ligand
Different allosteric enzymes conform to different models - the ATCase is in the middle (semi-sequential)
How are substrate analogues used to study reaction mechanisms and co-operativaty?
Substrate analogues
Molecules similar in structure to the reaction intermediate that can bind to the allosteric site and inhibiting the enzyme. Transition state analogues can be used to trap the enzyme in the transition state which it is normally only in for very brief periods.
E.g. PALA is a reaction intermediate for the ATCase. It differs slightly in structure and is not charged which makes it stable so will stay bound to active site whereas the substrate is charged and highly stable. It is a bi-substrate analogue as it has a similar structure to both substrates.
How is sit directed mutagenesis used to study reaction mechanisms and co-operativaty
By mutating residues in the active site to different amino acids their importance to the catalytic site and enzyme binding can be measured.
E.g. (ATCase)
His134 - Ala: kills activity - this confirms that it is important to the active site. The substitution is non conservative as the amino acids aren’t chemically similar
Lys-84 - Arg: Kills activity - the change is conservative but still kills activity which suggests the amino acid is critical and involved in the catalytic process
Lys-83 - Gln: Reduces activity - NO Cooperativity (no longer allosterically regulated)
Gln-133 - Ala: Enhanced cooperativity - this can sometimes happen
