Lecture 17 - Enzyme regulation II - covalent modification and compartmentalisation Flashcards
Describe the difference between allosteric regulation and covalent modification/compartmentalisiation
Enzyme regulation via covalent modification, compartmentalisation and aggregation/polymerisation all occur over a longer time scale (seconds-minutes) in comparison to allosteric regulation.
Name some types of post-translational modifications.
In addition to phosphorylation there are a number of possible alternative post-translational modifications:
* Methylation
* Uridylation
* Adenylation
* ADP-ribosylation
* Acetylation
Ubiquitination - The addition of ubiquitin molecules to a protein targets It for proteosome degradation.
What is the role of phosphorylation in enzyme regulation?
Phosphorylation
Phosphorylation is catalysed by kinases and describes the addition o f a phosphate to the hydroxyl group of Ser, Thr, or Tyr residues. (the addition of a bulky negatively charged group to a moderately polar residue)
1. Can block the active site - E.g. isocitrate dehydrogenase
2. Oxygen atoms can hydrogen bond with residues on the polypeptide
3. Negative charge on phosphate group can repel negatively charged (Asp, Glu) or attract positively charged residues (Lys, Arg)
Phosphorylation therefore often leads to conformational changes in the protein.
Phosphorylation is reversible and the modification can be reversed by phosphorylases
Phosphorylation can therefore activate or inactivate a protein.
Phosphorylation can lead to complex fine-tuning of metabolic regulation. There are a number of known phosphorylation sites on each subunit of glycogen synthase and depending on the site phosphorylated it will affect the activation/inactivation of the protein.
Phosphorylation recognition sequences are diverse and some kinases have broad whilst others have specific recognition sequences
How does phosphorylation lead to regulation of the enzyme glycogen phosphorylase?
The activation and inactivation of glycogen phosphorylase by phosphorylase kinase and phosphorylase phosphatase.
* Phosphorylase kinase phosphorylates the Ser-14 on the glycogen phosphorylase activating it. (The phosphate is from ATP which is converted into ADP). The enzyme is a dimeric enzymes so there are two Ser-14 phosphorylated and therefore 2 ATP needed.
Phosphorylase phosphatase removes the phosphates returning the enzyme to the tensed state where the affinity for the substrate is less.
How are the regulators of key metabolic enzymes regulated?
Protein kinase activity is often regulated in response to the change in second messenger concentration that is a response to hormone action ([Ca2+], [cAMP])
E.g. activation of the phosphorylase b kinase by PKA - PKA (protein kinase A) = cAMP dependent kinase
Displacement of an auto-inhibitory subunit underpins second messenger-dependent allosteric activation of numerous protein kinases.
Some hormones directly activate a protein kinase
E.g. Insulin dependent activation of its RTK
1. Insulin binds to a dimeric receptor (INSR)
2. Ligand occupation activates the tyrosine kinase domain of each β-subunit and autophosphorylation of Tyr residues in the C-terminus of the opposing subunit occurs
3. Autophosphorylation of receptor fully opens up the TYR-kinase active site of each subunit
4. Phosphorylation of target protein which effects
a. Metabolic enzymes
Gene expression
How do cells respond to a decrease in [ATP]?
The AMP activated protein kinase AMPK responds to changes AMP concentration which is a more sensitive indicator of the cells energetic state than [ATP].
A small drop in ATP concentration causes a large change in AMP concentration.
* AMPK in response to AMP up regulates catabolic processes and inhibits anabolic processes such as glycogen synthesis. This leads to an increase in ATP concentration. The ATP inhibits AMPK. This continues until an equilibrium is reached.
Trimeric mammalian AMPK is composed of 3 subunits.
* α-catalytic subunit
* β-adaptor subunit (holds trimer together)
* γ-subunit - contains AMP binding sites which regulate the kinase through allosteric regulation (activation)
AMP binding exposes the Threonine 172 residue on the α-subunit which means it can be phosphorylated by the upstream kinase kinase.
Activation of AMPK occurs by three independent mechanisms
1. Allosteric activation by AMP of phosphorylated enzyme
2. AMP dependent promotion of phosphorylation of Thr172 by an upstream kinase (the AMPKK aka LKB1)
3. Inhibition of the THR172 dephosphorylation
High [ATP] antagonises the binding of AMP, resulting in no activation
The three way mechanism of regulation allows for increased sensitivity so a small change in [AMP] causes a large change in activity of AMPK.
How does compartmentalisation regulate glucokinase?
Regulation of enzyme activity by dynamic changes in enzyme localisation (or compartmentalisation)
E.g. Glucokinase aka Hexokinase IV
Glucokinase is an isoform on hexokinase that is found in the liver
* It has no feedback inhibition by glucose-6-phophate
* Much higher Km form glucose than HK I-III
In the absence on glucose glucokinase binds a regulatory protein which sequesters the enzyme in the nucleus.
In the absence of glucose, the binding affinity of glucokinase for its regulatory protein is enhanced by the positive effector fructose-6-phosphate (therefore the liver does not compete with other organs for scarce amounts of glucose)
An increase in intracellular glucose concentration disrupts glucokinase-regulator interaction and glucokinase moves into the cytosol (Liver is poised to respond rapidly to increased blood glucose levels)
