Control of Metabolism Flashcards
(30 cards)
Glycolysis in active vs resting muscle
Hexokinase: G6P negative feedback
PFK1: ATP/AMP allosterically inhibits
F-1,6-BPase: AMP allosterically inhibits
(polarises substrate cycle)
Pyruvate kinase:
Inhibited byATP/acetylcoA/fatty-acids
Activated by F16BP
Glycogen Metabolism in Muscle: PKA
Phosphorylase kinase (aa and bb subunits - or by Ca2+ binding to d subunit, calmodulin)
Phosphorylase (b–>a)
Glycogen Synthase (by PKA and phos’ kinase)
PP-1 (G subunit and inhibitor protein by PKA)
- insulin phos’ on different site, activates PP1
Phosphorylase in Muscle
Inactive Phosphorylase b T-form in resting muscle
Converts to Phosphorylase a due to Ser14 phosphorylation; exists predominantly in active R-form - shifts a-helices and places Lys and Arg in active site, increasing affinity of Pi.
Dephosphorylated by PP1 back to Phosphorylase b,
Active Phosphorylase b ‘R-form’ stabilised by AMP, while ATP and G6P stabilises T-form
Glycogen synthase in muscle
Phosphorylation on 7 possible Ser residues increases Km for substrate (UDP-glucose) thus decreases activity
Phosphorylated by PKA and Phosphorylase kinase on the same residue
Decreases affinity for activator G6P and increases affinity for inhibitors ATP and Pi
PP1 in muscle
Reverses phosphorylation of substrates of PKA (including Ser14p on phosphorylase, and glycogen synthase).
Catalytic subunit has low affinity for glycogen particles
G subunit confers high affinity and draws PP1 into the glycogen particle. Phosphorylation of G subunit by PKA prevents it from binding catalytic subunit.
Phosphorylation of inhibitor protein by PKA inhibits catalytic subunit.
Insulin results in phosphorylation of G subunit on a different site that activates PP1.
Liver Glycogen Metabolism
Blood glucose high –> Phosphorylase a T-form stabilised –> Dephosphorylation of Ser14 by PP1 –> Release of PP1 –> Activates Glycogen Synthase
G6P promotes dephosphorylation and activation of glycogen synthase
AMPK - recognises ATP depletion and initiates metabolic response:
allosteric binding by AMP tethers it to PM, allowing phosphorylation by LKB1. Recognises ATP depletion, phosphorylates and inactivates glycogen synthesis.
Also mediates B-oxidation of fatty acids, inhibits fatty acid synthesis,
Glycolysis differences in liver vs muscle
Substrate-level control: GluT2 and Glucokinase
Co-operative binding of glucokinase
Glucokinase inhibited by regulatory protein rather than G6P
PFK-1 is allosterically regulated by F-2,6-BP
Liver isoform of pyruvate kinase can be phosphorylated
Liver gluconeogenesis by PKA activation
Activation of PKA INHIBITS GLYCOLYSIS and stimulates gluconeogenesis in the liver (c.f. muscle)
PFK-2/F-2,6-BP - phosphorylates a Ser residue, activates phosphatase and inhibits kinase, reducing F-2,6-BP. This decreases PFK-1 activity and increases F16BPase activity
Pyruvate kinase
Allosterically activated by F16BP, allosterically inhibited by ATP and alanine.
Phosphorylated by PKA and/or CAMK, inhibits its activity - most efficient when inhibited by ATP and alanine.
Gluconeogenesis in the liver by PKA-independent mechanisms
Low blood glucose - lowers GluT2 activity which lowers glucokinase activity (co-operative binding)
High glucose –> acetylation of PEPCK, promotes its degradation
Glucokinase
Inhibited by regulatory protein. F6P reinforces inhibition (negative feedback) F1P relieves inhibition.
Effects of insulin on liver
Promotes glycolysis, inhibits gluconeogenesis
Activates protein phosphatase - removes phosphorylation of PFK2-F26BPase, thus activating kinase and inhibiting phosphatase
Activates low Km cAMP phosphodiesterase
Transcriptional control of liver gluconeogenesis/glycolysis
Insulin stimulates expression of PFK-2/F26BPase and also activates its kinase activity
Stimulates expression of PFK-1 and pyruvate kinase
Glucagon activates PKA, which phosphorylates CREB.
CREB binds to CRE-elements, recruits p300/CBP, activates transcription of PGC1, PEPCK.
PGC-1 co-ordinately upregulates gluconeogenic enzymes, PEPCK, G6Pase, F-1,6-BPase
High glucose –> ChREBP is dephosphorylated by PP2A, binds to ChRE-elements –> Upregulates glycolytic genes (eg. pyruvate kinase)
Low glucose –> ChREBP is phosphorylated by PKA and AMPK –> Nuclear exclusion, reduces DNA-binding affinity
Regulation and activity of HIF-1
Degraded and inactivated in the presence of oxygen. Two Prolyl residues on HIF1 are hydroxylated - degraded by ubiquitination.
Hydroxylates Asn residues, which blocks its interaction with p300/CBP.
HIF-1 upregulates almost all glycolytic enzymes, and prevents entry into TCA cycle by inducing PDK1 expression, which phosphorylates and inactivates PDH
DIversion of glycolytic intermediates
p53 –> TIGAR, a F26BPase that inhibits glycolysis –> Diverts flux into PPP –> nucleotides for DNA repair and NADPH for protection against oxidative stress
PGAM1 converts 3PG to 2PG, promotes flux into serine biosynthesis pathway, upregulated in proliferating cells
p53 inhibits PGAM1 - so loss of p53 increases PGAM1 activity.
PKM2 in tumour cells inhibited by phosphotyrosine motifs, or oxidation of a Cys residue. Diverts flux into SBP, PPP
CyclinD-CDK6 phosphorylates PFK-1 and PKM-2, dissociates the tetramers into dimers, decreasing glycolytic flux.
Substrate channeling
Two sequential enzymes interact - so substrate never fully equilibriates with solvent.
in vitro isolated enzyme systems: GAPDH and phosphoglycerate - single functional complex where intermediate is never exposed to solvent
In vivo:
Hexokinase - brain cells (HK-I) vs muscle cells (HK-II, which associated with mt in response to insulin)
Uses intra-mitochondrially generated ATP
Cardiac cells - cell-permeable peptide with HKII-mitochondrial-binding motif - disrupts cardiac function
Aldolase and GAPDH by GPDH-1 in Drosophila flight muscle.
Endothelial cell motility: PFKFB3 localises to actin in cell cortex - bifunctional enzyme with higher kinase activity - favours glycolysis, supplies actin with ATP.
.
LOCALISATION: Creatine kinase
mt isoform associates with ANT and porins
cytosolic isoform associates with myofibrils
Temporal buffer and maintains locally high ATP:ADP ratios
Alternate diffusion pathway that is faster since [creatine] is much higher than ADP.
LOCALISATION of glycogen synthase and glucokinase in liver, and glycogen synthase in muscle
High glucose:
Glycogen synthase co-localises with actin in the cell cortex. Glucokinase translocates from the nucleus to the periphery, where it co-localises with glycogen synthase.
Low glucose:
Glucokinase sequestered in nucleus by GKRP.
GK makes G6P, which activates GS and promotes its dephosphorylation.
Skeletal muscle: Dephosphorylation of GS allows it to translocate into glycogen particle to synthesise glycogen
LOCALISATION of BaD
High glucose –> Bad is phosphorylated and sequestered near mitochondria. Phosphorylated Bad stimulates Glucokinase.
low glucose –> Bad is activated, triggers apoptosis
PI3K effects
PI3K activated by growth factor signals and insulin.
PI3K activates Rac, which mobilises aldolase from actin, promotes glycolytic flux.
Activates Akt2:
Phosphorylates hexokinase, promoting association with mt.
Phosphorylates and activates PFK-2, activating PFK-1
Causes exocytosis of GluT4-vesicles, promotes GluT4 insertion.
Scale free network
Small number of metabolites that are highly connected and conserved
Any two metabolites are connected by a short path
How to identify flux control points
Low Vmax
Mass action ratio is far from Km
Cross-over points (increase in flux is accompanied by decrease in substrate and increase in product)
Control points (immediately after branch-points)
Purpose of flux control
Fulfil changes in demand
Prevent futile cycling
Control flux into other pathways
Methods to control flux
Control expression of an enzyme
Control activity of the enzyme
Control of intermediates (substrate cycling, diverting flux)
Flux control coefficients examples
ANT has high FCC when rates of respiration is high (titrate activity using carboxytractyloside)
Citrate synthase has high FCC for TCA cycle on acetate medium but not glucose - titrate expression under trp/lac promoter using IPTG
Glucokinase has high FCC for blood glucose regulation in transgenic mice expressing GK under an inducible promoter (high blood glucose, impaired glycogen synthesis)
Fluorescence microscopy
Wide-field microscopy - weak z resolution
confocal microscopy - selects for certain z focal plane, thus defining a slice of the sample
multi-photon microscopy
Light sheet microscopy
