Baines Flashcards
Factors that determine the activity of enzymes (9)
Extra cellular signals Transcription of genes mRNA degradation mRNA translation Protein degradation (ubiquitin) Sub cellular organelle Enzyme binds substrate Enzyme binds ligand Enzyme phosphorylated/ de phosphorylated
Define homeostasis
Adequate supply of metabolites for normal function. Substrate is provided by preceding reaction at same time it is turned into product. Adaptations to wound healing, fight/flight
Post translational modifications of enzymes
Phosphorylated on Ser, Thr, Tyr by kinases.
Acetylation of Lys, hydroxylation of Pro, o-glycosylation of Ser.
Kinase + ATP -> phosphorylation
Phosphoprotein Phosphatase +H2O -> dephosphorylation
Protein kinase A
cAMP dependent kinase
Hormone -> GPCR -> adenylate cyclase -> cAMP
Inactive: R unit inserts inhibitor sequence into C unit
Active: 2 cAMP bind to each C unit. Binding releases C units, activates.
Phosphorylates.
Pyruvate kinase isoforms
Regulation
Liver- N terminal extension. Arg-Arg-X-Ser
Substrate for PKA which phosphorylates Ser
Glucagon -> cAMP -> PKA -> serP -> inactive pyruvate kinase
Stops PEP -> Pyruvate
Stops glucose consumption when low levels
Activated by high F16BP (promotes glycolysis)
Short range control
The 3 irreversible steps of glycolysis
3 steps - hexokinase -PFK1 -Pyruvate kinase AMP activates, inhibits FBPase Intermediates of Krebs inhibit G6P inhibits HK- ensures that made by Gluconeogenesis is not consumed in futile cycle.
ATP and AMP as cellular regulators
Equation of PFK1
Citrate as regulator
When ATP -10%, AMP x600%
ATP +F6P –> ADP + F1,6BP
Citrate increases the inhibitory effect of ATP
Fructose 2,6 BP
Additional mechanisms needed for the liver
Activates PFK1, increases affinity for F6P (binds to R site)
Inhibits fructose-1,6-bisphosphatase
PFK2 function
Creates F2,6BP and ADP –> increases PK1 affinity for F6P
Reversed by FBPase2
Both regulated by insulin and glucagon
FBPase inhibited by F2,6BP, more sensitive to AMP inhibition.
Hormonal regulation of PFK1 by glucagon and insulin
Glucagon -> cAMP -> PKA -> PK2 phospharylated and inhibited.
Insulin -> Phosphoprotein phosphatase -> activates PK2
Insulin -> PK2 -> F26BP -> activates PFK1 -> glycolysis
Glucagon -> xPK2 -> less PK1 -> Gluconeogenesis
AMPK (AMP activated protein kinase)
ATP –> ADP
ADP + adenylate kinase –> AMP
AMPK is activated by AMP
Shifts from energy consuming to energy releasing
Crystal structure of AMPK
Thr172 can be phosphorylated which activates
B subunit can bind glycogen
Y subunit has 4 CBS1 sites where AMP binds.
Control of glycolysis in the heart
Rapid ATP turnover Adenylate kinase converts ADP -> AMP AMP activates AMPK AMPK phosphorylates PFK2 PFK2 creates F26BP F26BP activates PFK1 More F6P --> F16BP for glycolysis
The activation of AMPK
Can be activated by at least 3 kinases
Calmodulin- dependent protein kinase (CaMKKβ)
Calmodulin has 4 Ca binding sites. Once activated by Ca, spreads by autophosphorylation (no more Ca needed).
How does Adenosine inhibit glycolysis?
During low energy supply/ ischemia it accumulates.
Inhibits glycolysis by acting on AMPK, PFK2 and PFK1
Limits cardiomyocyte injury
How is calcium overload created?
Blockage -> hypoxia -> AMP -> AMPK -> glycolysis and eNOS
ATP is restored, but there is acidosis which gives Ca2+ overload.
The role of eNOS and adenosine in stopping calcium overload
eNOS -> NO Causes vasodilation and restores O2 Adenosine in the blood activates PP2A -> inhibits AMPK Glycolysis slowed, acidosis reduced. Ca load reduced. Homeostasis
How does AMPK enhance NO availability?
Lack of O2 –> activates AMPK
AMPK –> phosphorylates eNOS (complexed with HSP90)
eNOS converts Arg + NADPH + H + O2 –> citrulline + NO + NADP
AMPK inhibits the removal of NO –> ONOO radicals
How does NO cause vasodilation?
Activates guanylate cyclase
- increases cGMP which stops Ca entry and lowers levels
- Activates K channels, relaxation
- cGMP protein kinase
- cGMP PK activates myosin light chain phosphatase
- smooth muscle relaxation
How does AMPK inhibit hepatic Gluconeogenesis (CRTC2)
Transcriptional control of Gluconeogenesis by CRTC2 and AMPK.
CRTC2 –> CREB –> PGC1α, HNF4α, FOX01.
These control expression of PEPCK and G6Pase
LKB1 activates AMPK –> phosphorylates CRTC2 -> out of nucleus
The release of CRTC2 stops Gluconeogenesis via CREB.
AMPK also phosphorylates HNF4α –> inhibits transcription factors for Gluconeogenesis enzymes
Definition of flux
Key enzymes far from equilibrium, regulation points.
Net = Vf - Vr
Later products act as flux inhibitors
Mass action ratio (Q) - product:reactant
Rate limiting step
Flux cannot be faster than the slowest step.
More than one enzyme could regulate this step.
Metabolic control analysis
Vary the amount of an enzyme and see effect on flux.
Pathway has value of 1, enzyme has control coefficient of 0-1.
Side reactions will have -ve flux.
Double-log plot, is the gradient of tangent.
Flux control coefficient
Relative contribution of each enzyme to setting the rate of flux.
Side reactions are -ve.
