block 7-regulation of protein function Flashcards
(35 cards)
Why are proteins regulated
- Maintenance of cell homeostasis
- Responsiveness to the environment
- Efficiency
Major ways in which enzyme activity can be regulated
- Changes in substrate concentration
- Role of isoenzymes
- Binding of small effector molecules
- Allosteric regulation
- Reversible covalent modification
- Phosphorylation
- Binding of regulatory proteins
- Proteolytic activation
- Controlling the amount of enzyme present
Changes in substrate concentration
Changing the substrate
concentration will affect the rate of
an enzyme catalysed reaction
Particularly important at [S] less
than Km where the rate is linearly
dependent on [S]
Some substrates will have limited
availability
e.g. total cellular concentration of
NAD+ + NADH is constant but ratio
of the two coenzymes can vary
what are isozymes?
Enzymes that catalyse the same reaction but have a different amino acid
sequence
* Can be encoded by different genes or derived by alternative
splicing from one gene
* Have different kinetic/regulatory properties
what role do isosymes play?
Different metabolic uses in different organs
Different locations and metabolic roles in the same cell
Different roles at different stages in development
Different responses to allosteric regulators
lacatate dehyrogenase
-5 different isozymes
-each contain 4 polypeptides composed of two different types of proteins
main forms: H4= mainly in heart, low km for lactate(favours lactate oxidation)
-m4= mainly in skeletal muscle
-low km for pyruvate (favours pyruvate reduction)
allosteric regulation
Defined as regulation mediated by interactions of a modulator at a
regulatory site away from the active/binding site
* Binding of the modulator causes a change in conformation that affects the
activity of the enzyme
* Allosterically regulated enzymes are usually multi-subunit proteins made of multiple subunits
* The modulator may be the same as the substrate – homotropic modulator(normal ligand/substrate for that protein) – or
different – heterotropic modulator (not normally the substrate)
* Regulatory sites may be on different subunits from the active site
why doesnt alosterically regulated enzymes obey michaelis-menten kinetics?
They show a sigmoidal (S-shaped) curve instead of a hyperbolic one.
This is due to a change in substrate binding affinity.
The enzyme switches between a low-affinity T state (tense) and a high-affinity R state (relaxed).
cooperative oxygen binding in haemoglobin
- sigmodal binding curve because it has 4 different subunits and switches between states
-Deoxyhaemoglobin can exist in low affinity T state or high affinity R state
Oxygen binding promotes stabilisation of the R state=shift the equilibrium to the high affinity stae= conformational shape occurs= breaking electrostatic interactions allow haemoglobin to move more freely
-low affinity state can bind oxygen but more likely to be in the deoxygenated form. - make sure you study the shift in equilibiiums at different enzymes/km etc…
allosteric enzymes
Allosteric activators - Increase the proportion of enzyme in the R state
Allosteric inhibitors - Increase the proportion of enzyme in the T state
covalent modification
- Covalent modifications are made post-translationally, after the protein is formed
- Most modifications are reversible
- Usually mediated by the action of enzymes
- Multiple covalent modifications on the same protein allow for complex regulation of
protein activity - binding on a group to a covalent interaction e.g. phosphorylation, acetylation etc..
phosphorylation
-includes two enyzmes
Protein kinases= transfer the terminal phosphate from ATP to the –OH group of Ser, Thr, Tyr
Protein phosphatases =reverse the effects of kinases by catalysing the hydrolytic removal of phosphoryl
groups from proteins.
why is phosphorylation effective?
- Large free energy of phosphorylation
provides energy to allow a shift in the conformation of the phosphorylated protein - Addition of 2 negative charges
disrupts existing electrostatic interactions and may allow new ones to form - A phosphoryl group can form three or more hydrogen bonds
allows for specific interactions with new hydrogen bond donors - Kinetics of phosphorylation can be adjusted
may be from a few seconds to a several hours - Allows amplification cascades
- ATP is the cellular energy currency
What effect can protein phosphorylation have?
- some enzyme activity in activity and some is inhibited
-Phosphorylation affects the
structure of the molecule - allosteric activation
-can changes enzymes to the r-state or t- state
how do the protein kinases know which ser,thr,thy groups to target?
Each protein kinase has its own consensus sequence.
This sequence determines the specific target sites on the protein.
Specificity ensures that the kinase targets only certain residues for phosphorylation.
go back to answer lecture questions
make sure you fully understand
how are protein kinases activates?
The binding of small molecules/proteins causes activation of protein kinases
e.g. camp, Ca2+, calmodulin
-
amplification by kinase cascades
Kinase cascades involve a series of protein kinases activating each other in sequence.
This allows the initial signal (e.g., from a receptor) to be amplified many times over.
The result is that a small signal can produce a large cellular response very quickly (in just milliseconds).
This process increases the strength and speed of the cellular response to external signals.
activation of protein kinase A
Activation through G-protein
coupled receptors
(GPCR)
Gs activates adenylyl cyclase
-PKA Structure:
Regulatory subunit (R): 49 kDa
Catalytic subunit (C): 38 kDa
Together, they form a R2C2 complex when there is no cAMP.
Regulation:
In the absence of cAMP, the R subunit binds to the C subunit, blocking its activity and preventing phosphorylation.
Activation:
Binding of cAMP to the R subunit causes a conformational change, releasing the C subunit and activating its kinase activity.
Consensus sequence:
PKA’s target sequence is R - R - X - S - I (where X is any amino acid).
Pseudosubstrate sequence:
A specific inhibitory sequence (like R - R - G - A - I) that mimics the substrate but doesn’t get phosphorylated, helping to regulate PKA’s activity.
regulation of proteins by calcium
-Activates calmodulin
Calmodulin is a small protein (17 kDa) that binds calcium ions (Ca²⁺).
It belongs to the EF-hand protein family, which means it has a special structure for binding calcium.
Each EF-hand has two α-helices connected by a short loop where the calcium binds.
Each Ca²⁺ ion is coordinated by 7 oxygen atoms, helping calmodulin to tightly and specifically bind calcium.
When calmodulin binds calcium, it changes shape, which allows it to interact with and regulate other proteins.
-know breifly
go back to lecture questions too
regualation by partial proteolysis
-Proteins can be synthesised in
longer inactive forms –
proprotein or zymogen
-Removal of pro-segment by
breaking a peptide bond
releases the fully active form
why are mechanisms regulated by partial proteolysis
- Highly specific process – breaking of peptide bond in the zymogen catalysed by an enzyme
- Irreversible
- Cleavage does not need ATP
- Can be used to activate intracellular and extracellular proteins e.g. activation of trypsin or chymotryosin
How are activated zymogens switched off?
-Tight binding of inhibitors
-Degradation
