Control Mechanisms Flashcards
Course (coarse??) control?
Amount of enzyme present based on synthesis/degradation - slow
Fine control?
Activity of enzyme e.g. phos status, level in cell, substrate availability; rapid, can take ms
Passive control and MM kinetics?
Where at low S, V
Features of irreversible inhibitors and example?
Covalent modification
Often at active site, blocking S
Toxic
e.g. diisoprrpylphosphofluoridate, prototype of serine nerve gas which modifies a serine in the active site of acetylcholineesterase blocking action potentials
Reversible competition example?
Succinate dehydrogenase in citric acid cycle
Succinate – fumurate, inhibited by malonate
Can be overcome by high S
MM kinetics of each inhibition?
Irreversible: ? like removing the enzyme
Competitive reversible - Vmax same, Km up
Non-competitive reversible: Km same, Vmax down
Non-competitive reversible inhibition example?
Enzyme has both active and inhibitor sites, where I may prevent activity of S (without stopping it binding)
e.g. F16BP, gluconeogenesis by ATP
Feedback regulation in linear pathways?
End product controls rate of production
Avoids intermediate build ip
Stops unproductive over-production
Often reversible
Feedback regulation in branched pathways? (2)
2 or more end products needed in different amount, uses sequential feedback inhibition or nested feedback inhibition
Sequential feedback inhibition?
End product regulate intermediate step, often at branch point to cause linear inhibition of earlier bit
e.g. DAHP synthase in aromatic AA synthesis
Nested feedback inhibition
Both end products inhibit the first step, only for single regulatory enzymes with multiple inhibitor binding sites
e.g. purine biosynthesis
Control of mechanisms with multiple enzymes?
Nested feedback inhibition
Isoenzymes
What differs between isoenzymes?
Km
Cofactor requirements
Localisation
Genetic encoding
Examples of isoenzymes
Aspartokinase
Hexokinase
Allosteric regulators are all what?
Multi-subunit proteins, with multiple active sites
Homoallostery?
Co-operative substrate binding; 1 substrate (the ‘primer’) induces a conformational change to impact future binding potential
Gives sigmoidal kinetics e.g. haemoglobin
Heteroallostery?
Other effector molecules (not substrates) affect enzyme activity, either activating (stabilise active form) or inhibitors (stabilise inactive)
Regulation within a narrow S concentration range
Covalent modifications? ^)
Need energy; often in signalling pathways
Acetylation (of lysines)
Methylation (glutamate/aspartate)
Nucelotidylation (tyrosines)
ADP ribosylation (arginines)
Phosphorylation (OH group of serine, threonine, tyrosine)
Phosphorylation? Effects (4)?
Kinases and phosphates use phosphate group from ATP Ser + Thr - one class Tyr - second class Changes hydrogen bonds, negatively charges, affects 3D structure and S binding/catalysis
How do kinases recognise the residue?
Through consensus sequences - so as to avoid phosphorylating every AA that occurs
Nucleotidylation?
Addition of AMP = adenylation
UMP - uridylation
Used in biogenesis of organic nitrogen
First step in biogenesis of organic nitrogen?
Ammonia is assimilated into one of three pathways: carbamoyl phosphate, aspartate, or glutamate
Glutamate - glutamine regulation?
- Glutamate dehydrogenase catalyses reductive amination of alpha-ketoglutarate to form glutamate
- Second ammonia added to glutamate - glutamine via glutamine synthetase
Regulation of glutamine synthetase (GS)?
2 stacked rings of 6 subunits with 8 binding sites each for each inhibitor, of which all 8 must be present. (=96 effector sites in total)
Adenylation of Tyr397 near active site by AMP addition, forming an ester bond
What catalyses adenylation of GS?
Adenyl transferase and PII (regulatory protein), which in itself is regulated by uridylylation.
PII-UMP = deadenylation = activation
What happens when nitrogen accumulates?
Glutamine inhibits uridylylation of PII, deadenylation of GS stops, adenylation continues i.e. build up of inactive AMP-GS, glutamine synthesis stops
Stable proteins? (i.e. long half-lives)
Histones, haemoglobin, crystallin
Often structural, with constant catalytic activity
Unstable proteins?
HMG-CoA reductase (2hrs), ornithine decarboxylase (11 mins)
Often regulatory, TFs, or catalyse committed steps
Pathways of degradation?
Protease-mediated
Lysosomal
Ubiquitin-proteosome
Protease mediated degradation?
Intestinal; GI tract, stomach, intestine
Degrades exogenous dietary proteins
Lysosomal degradation?
Ingested material/obsolete cell components in bulk
Influenced by nutrients/growth factors e.g. in starvation
Contain hydrolases in an acidic environment maintained by a proton pump - in the neutral cytosol, hydrolases do not function
Pathways of lysosomal degradation?
Endocytosis - extracellular proteins are packed in early endosomes to form late endosomes for lysosmal delivery
Autophagy - cellular proteins/complete organelles like mitochondria. Autophagosomes form around them and bring to lysosomes
Is autophagy selective?
No - it is not regulated in healthy cells, occurring at a rate of about 1-10% of total cell protein per hour controlled by delivery to lysosome
Where is the Ub-proteasome system (UPS) used?
Regulation of short lived proteins
Removal of unwanted proteins
Removal of mutant/damaged proteins
Ubiquitination?
Ubiquitin = 76AA, binding proteins with an isopeptide bond at its C-terminal glycine. Polymerisation = signal for degradation
UPS genes?
E1s - 1-2 activating enzymes,
E2s - 10-20 conjugating enzymes
E3s - 500-800 ubiquitin ligases, catalysing final transfer to sustrate
N-end rule?
Regulates half-life of cytosolic proteins, where specific AAs make up degradation motifs recognised by E3s e.g. arginine, isoleucine, leucine
PEST sequences?
Proline, glutamate, serine and threonine-rich regions direct degradation through their phosphorylation sites, where a protein kinase allows E3s to recognise them
