L4 - Allosteric regulation of enzymes Flashcards
Allosteric: what are the origins of the word, what molecules have allosteric parts, what are allosteric effectors usually, and what can allosteric effectors do?
Allos - other
steros - shape
Multi-subunit proteins with multiple active sites.
Usually small chemicals
- Allosteric effectors can be positive, or improve enzymatic catalysis
- Allosteric effectors can be negative, or reduce enzymatic catalysis
Homoallostery
Co-operative substrate binding
Heteroallostery
Regulation by effector molecules
May either stabilise the active form (activator) or stabilise the inactive form (inhibitor)
Co-operative binding: what does it do, how does it work, and what kind of graph shape does it show?
Can amplify enzyme activity
- Substrate binding at one site stabilises other favourable conformational changes at other subunits - primes the enzyme to accept additional substrate molecules more readily
Sigmoidal-kinetic curve - S-shaped curve
Allosteric regulators: do they follow Michaelis menten kinetics?
The kinetics of allosteric regulators differ from Michaelis-Menten kinetics
Aspartate carbamoyltransferase: what does it do, how is it regulated, and what is it an example?
Converts aspartate into pyrimidine
Allosteric regulation
“Commitment” step - once this reaction occurs, aspartate can no longer be used in protein synthesis and is ‘committed’ to this process
Covalent modification: what does it do and what examples of them are there?
Reversible/irreversible regulation of enzyme function requiring expenditure of energy - often used in signalling
- Acetylation of lysine or amino-terminal groups
- Methylation of glutamate or aspartate residues
- Nucleotidylation of tyrosine residues
- ADP ribosylation primarily of arginine residues
- Phosphorylation major covalent modification
Kinases and phosphatases: what are the main classes?
- Specificity for Ser and Thr residues
- Specificity for Tyr residues
Effects of phosphorylation: what amino acids are affected, what modifications does phosphorylation cause to occur, what are consensus sequences and what do they mean for phosphorylation?
Ser, Thr, Tyr - moderately polar
- Bulky charged group added
- Potential hydrogen bonds can be formed
- Negative charge
- Three-dimensional structure - enzyme conformation, substrate binding and catalysis
Consensus sequences - sequences that determine whether a protein will be phosphorylated, allows specificity by making it so that not all proteins get phosphorylated
Glycogen phosphorylase: what is it, what does it do, what is the process of its (de)phosphorylation, and what are two allosteric effectors?
Dimer with 2 interconvertible forms:
* Active - phosphorylase a
* Inactive - phosphorylase b
Converts Glycogen into Glucose-1-phosphate, mobilising the energy source
Kinases/phosphatases recognise a sequence - Serine-14 and (de)phosphorylate it (in)activate it
AMP - low ATP, high AMP means the cell needs more energy
Epinephrine - action needed, energy required
The diagram illustrates a common post-translational modification which can be used to regulate the activity of key metabolic enzymes.
What is the name of the class of enzymes that catalyse this modification [1 mark]
Which three amino acids can undergo this modification [1 mark]
Explain three possible mechanisms by which this post-translational modification can alter enzyme activity [3 marks]
Kinases
- Threonine
- Serine
- Tyrosine
- Bulky charged group added
- Potential hydrogen bonds can be formed
- Negative charge
- Three-dimensional structure - enzyme conformation, substrate binding and catalysis
Nucleotidylation: what is it, what are the two forms of it, and what can it be used for?
Covalent modification using nucleotide bases
Addition of:
AMP - adenylation
UMP - uridylation
Biogenesis of organic nitrogen/assimilation of ammonia (NH₃):
* Carbamoyl Phosphate -> Arg -> Pyrimidines -> Urea
* Aspartate
* Glu -> Gln -> Purines, Trp, His, Nucleotides, Amino sugars
Glutamate dehydrogenase: what is it, what does it do, how does it do it, is it reversible, and what are its allosteric effectors?
An enzyme that is used to incorporate ammonia into organic matter
Reductive amination of α-ketoglutarate: using NADPH + H⁺ to react with 2C’s C=O to form H₂O + NAPH⁺ and result in the binding of NH₃ to α-KG
Reversible - can both assimilate ammonia and generate α-KG for the citric acid cycle
- Inhibited by ATP or GTP
- Stimulated by ADP or GDP
Glutamine synthetase: what does it do, what is the process that allows this, is it reversible, why is it tightly regulated, what are its effectors, and how many effector sites does it have?
Converts glutamate into glutamine using ATP
Using ATP, NH₃ reacts with glutamate and replaces the terminal -OH group on the glutamate, forming an NH₂ bond and producing H₂O
Since it requires ATP, it is irreversible
- Biosynthesis of A.A.s
- Purines and pyrimidines
- Detoxification of ammonia
- Supply of a-KG
Covalent modification and allosteric regulation – cumulative feedback inhibition:
Eight specific inhibitors bind to different allosteric sites and inactivate the enzyme when all are bound:
* Glycine
* Alanine
* Glucosamine-6-phosphate
* CTP
* Histidine
* Carbamoyl phosphate
* Tryptophan
* AMP
Eight inhibitors, 12 subunits = 96 effector sites
Nucleotidylation of glutamine synthase: how does it affect it, what does it do, and what occurs as subunits get inactivated?
Adenylation at a specific Tyr residue (Tyr-397, near active site)
AMP is transferred from ATP - inactivating it
From 1 through 12, the monomers in the holoenzyme can be modified, causing progressive inactivation as the ratio of [modified]/[unmodified] GS subunits increases
