Enzyme Regulation Flashcards
Feedback inhibited enzymes
Excessive build up of end products inhibits enzyme activity
Covalently modified enzymes
Reversible attachment of small molecules that inhibit activity (phosphorylation, methylation and ribosylation)
Allosteric enzymes
Conformational changes by reversible non covalent binding of co-factors and regulatory sub-units
Zymogens and proenzymes
Become activated by proteolytic cleavage of inactive precursor molecules
Enzyme specificity- Absolue
Defined to one substrate in its one define stereoisomeric state
Enzymes specificity- Group
Enzymes can recognise compounds belonging to a group e.g phosphates and kinases
Enzyme specificity- Promiscuity
Enzymes that can act upon several substrates regulation is very important
Specific inhibition is hard
Allosteric Regulation
Causes changes (inhibitor or activator) to active site so enzyme can bind or not bind
Homotropic regulation
The substrate and regulator bind to the same site
regulatory site=active site
Heterotrophic regulation
The regulator binds to site other than the active site
Co-operative regulation
Binding of the first substrate molecule to the active site makes it easier for more substrate to bind
Test for co-operability
The hill plot can be used to estimate the degree of cooperation between subunits
Log(theta/1-theta) = nlog [S]- nlogKA
Theta = fraction of bound enzyme [S] = ligand concentration KA= [S] at 50% occupation
Aspartate Transcarbamoylase
Catalyses the first step in pyrimidines
Precisely regulated to produce the right amount of CTP
Negativity allosterically regulated by CTP
T state (less active)= favoured by CTP
R state (more active)= favoured by substrate binding
Apo-enzyme
Just protein
Holo-enzyme
Protein and co-factor or co-enzyme
Cofactors and Coenzymes
Many enzymes require prosthetic groups that are non amino acid in nature
Where do the Co’s come from
Most cannot be synthesised
A precursor molecule must come from the diet
Co-enzymes
Organic: Carry groups between enzymes
Most are vitamin derived
Co-factors
Inorganic- Metal ions
Required for enzyme activity
Co-enzymes and Co-substrates
Co-enzymes often donate chemical groups such as C, H and NH2
These are consumed by the reaction and are hence called Co-substrates
Common co-enzymes
Biotin- carboxylation
Coenzyme A - Acyl transfers
FAD and NAD- Oxidation and Reduction
Aspartate Transcarbamoylase continued
6 regulatory and 6 catalytic subunits
Catalytic sites show normal MM kinetics and are unresponsive to CTP
Regulatory subunits have no catalytic ability but binds to CTP
CTP binding
Stabilises the T state
Inhibition changes the quaternary structure which makes is harder for substrate to bind
More substrate is required to achieve reaction rate
ATP and aspartate carbamolylase
Positive allosteric effector
Competes with CTP for site (alleviating inhibition)
Makes substrate binding easier and shifts equilibrium to the R state
Two reasons:
Signals high energy for mRNA synthesis
Maintaining the balance or pyrimidine and purine rings
Protein kinase A
Key name in the modulation of metabolic enzymes by covalent modification
Phosphorylates proteins
Each regulatory sub-unit (R) has a segment that is a pseudo-substrate for the catalytic Subunits
A rise in cAMP = binds to R and changes PKA shape = C subunits phosphorylates proteins
Phosphorylation
Most common reversible modification
Adds two negative charges which alters electrostatic interactions
Hexo-kinase is an enzyme that catalyses phosphorylation
Reversed by phosphatases
Glycogen Phosphorylase
Inter conversion between a and b forms of the enzymes adds or removes phosphate to the serine residue of to control its activity
Phosphatase and kinase respond to needs of cell
Phosphorylation at serine in response to epinephrine destabilises the N-terminal segment from acidic to basic environment
Allosterically activated by AMP(shows ATP levels are low) non covalently
Addition of glucose exposes the serine residue (phosphorylated) to phosphatase which converts the enzyme to inactive b-form
Riboflavin cofactors
Riboflavin phosphate
Flavin adenine dinucleotide (FAD)
Involved in metabolism of carbs, fats and proteins.
Hydrogen carriers in respiratory chain
Covalent regulation
Enzymes can be modified to change activity
Covalent regulation = reversible and sensitive
*can change from 0% to 100% activity
Phosphorylation (pyruvate dehydrogenase)
Adenylation (glutamate synthase)
Activation by proteolysis
Most proteases are synthesised as larger molecules, during the activation phase the inhibitory segment is cleaved
Activation may be after the protease is delivered to the compartment within the cell
Serine protease inhibitors
SERPINS
Proteins that block or enter protease active site to prevent substrate access
After the serpin is cleaved but prior to hydrolysis of of acyl-enzyme intermediate
The serpin undergoes S to R transition, causing the protease to go from the top to the bottom of the serpin the acyl-enzyme is hydrolysed really slowly and remains covalently attached and inhibited
SERPIN cleavage
Since the serpin must be cleaved
It becomes consumed and is therefore
Irreversible enzyme inhibitor
PEST proteins
Rich in
Pro (P)
Glu (U)
Ser (S)
Thr (T)
More rapidly degraded than other proteins
They have phosphorylation sites that allow them to be targeted by ubiquitin
Accelerated degradation
Proteins are turned over at a rate indicated by their half life
E.g haemoglobin 1/2 life = 120 days
But if an artificial amino acid is incorporated it can be degraded in 10 minutes
Degradation mechanisms
Non selective
- active proteases
- released in cells or present in lysosomes
- lysosomes usually degrade membrane proteins
Selective
- UPS (Ubiquitin proteome system)
- ATP dependent system requiring proteins to be tagged with ubiquitin for delivery to proteasome
Regulation of glycolysis
Controlled by:
Feedback inhibition
Allosteric regulation
Phosphorylation
