Control of Metabolism Flashcards
how is the atp pool buffered during intense exercise
by PCr
powering muscle contraction with glucose
- ATP from glycolysis
- intense exercise - PCr buffers ATP pool (though PCr is limited so must also mobilise glycogen
- anaerobic metabolism producing lactate - possible for short periods only
- prolonged exercise - aerobic metabolism of glucose, producing ATP more slowly, by OXPHOS
liver and blood glucose
- after a meal - insulin stimulates uptake of glucose, which is converted to glycogen, FAs, used as fuel
- after a fast - glucagon stimulates glycogenolysis, glucose release, mobilisation of FAs from adipose
3 key control points of glycolysis
hexokinase, PFK1, pyruvate kinase - irreversible steps with larger changes in gibbs free energy
what happens to lactate and alanine from anaerobic metabolism in muscle
released into blood and converted into glucose in the liver - glucose then released into blood and transported to muscle
= the cori cycle
6 ways to control enzyme activity
- substrate level control
- cooperativity
- allosteric regulation
- covalent modification
- substrate cycling
- control through changes in enzyme concentration
substrate level control
when most useful?
- enzyme has greatest sensitivity to changes in [S] when [S}
example of substrate level control
glucokinase - an isoform of hexokinase with a high Km for glucose
- B cell - glucokinase is glucose sensor, increase glycolytic flux and insulin release
- liver - glucose uptake
cooperativity definition
binding of first substrate affects binding of subsequent
increases sensitivity
concerted model of cooperativity
binding of first substrate shifts equilibrium in favour of relaxed form which has higher affinity for further substrate
sequential model of cooperativity
binding of first substrate causes conformational change from T to R (higher aff), this causes second subunit to change from T to R, facilitates binding of 2nd substrate
hill coefficient
protein p with n binding sites for ligand L
n=hill coefficient, a measure of cooperativity (< no binding sites)
(read eqn)
allosteric effectors
bind to a site on the enzyme other than the substrate binding site and regulate enzyme activity
eg activator could produce a conformational change to stabilise the R state
allosteric effectors in muscle glycolysis
- G6P allosterically inhibits muscle hexokinase
- PFK1 allosterically inhibited by ATP and activated by AMP
- PK allosterically activated by F16BP
sensitivity of … substrate level control, cooperativity, allostery
substrate level = linear response
others are more sensitive, sigmoidal rather than hyperbolic response curves
substrate cycling example
F6P, F16BP, via PFK1 and F16BPase
AMP allosterically activates PFK and inhibits F16BPase, allowing massive changes in flux with small changes in AMP levels/ the enzyme activities
- payed for by ATP, energy released as heat
types of covalent modification
phosphorylation
acetylation
shape of curve with cooperativity
sigmoidal
example of cooperativity in a monomeric enzyme
glucokinase = monomeric with 1 glucose binding site. has 2 slowly interconverting forms
- at low S, the low affinity E’ form dominates
- at high S, the high affinity E form dominates
this gives a sigmoidal saturation curve
AMP changes during exercise and allostery
- large increase in AMP during exercise as 2ADP==>ATP+AMP by adenylate kinase
- PFK1 activated by AMP, increases F16BP steady state
- increases allosteric act of PK
- decrease in steady state G6P, less inhibition of hexokinase
so a coordinated increase in activities of these enzymes
protein phosphorylation
- at OH groups of ser, thy, tyr
- introduces neg charge so can cause large conformational change
- by kinases, removed by phosphatases
these enzymes are promiscuous - have many targets. also many regulatory subunits
enzymes of glycogen metabolism
glycogen synthase converts UDPG to glycogen
phosphorylase converts glycogen to G1P, which is in equilibrium with G6P
how is gluconeogenesis like the reverse of glycolysis?
most of the reactions are at equilibrium so readily reversible for gluconeogenesis
- the HK, PFK1 and PK reactions are not (these ones use ATP) so different enzymes are required for the reversal:
F16BPase and pyruvate carboxylase+phosphoenolpyruvate carboxykinase
glycogen synthase and phosphorylase activities
don’t want simultaneous activity as will just turn over ATP
Phosphorylase
- AMP or phosphorylation converts to more active form (R form - allo, phosphorylase b->a - covalent)
- negative phosphate interacts with positive arginines, causing a large conformational change of a helices. exposes more + args of active site which are able to bind the negative phosphate of the substrate
lysine acetylation
almost every metabolic enzyme of the cell is acetylated - widespread importance in control
T,R, b,a forms of enzyme
T/R to do with allosteric regulation
a/b to do with activation by phosphorylation
adrenaline cascade and action on phosphorylase and glycogen synthase
- adrenaline activated adenylate cyclase
- cAMP
- cAMP activates PKA
- PKA activates phosphorylase kinase and inactivates glycogen synthase (-> b form)
- phosphorylase kinase phosphorylases and activates phosphorylase (-> a form)
phosphorylase kinase
- activated by phosphorylation by PKA
- activates phosphorylase and inactivates glycogen synthase by phosphorylation
protein phosphatase 1
reverses phosphorylation of phosphorylase, glycogen synthase, to decrease the rate of glycogen breakdown. GS and P are within the glycogen particle
- PP1 doesn’t have affinity for the glycogen particle until it associates with a G subunit
- PKA phosphorylates the G subunit, inactivating it (ensuring proteins remain phosphorylated, by turning off the dephosphorylation)
- phosphorylation of the inhibitor of PP1 activates the inhibitor, so inhibits the phosphatase
glycogen synthase and phosphorylase activation states
GS=active when dephosphorylated
phosphorylase is inactive when dephosphorylated
insulin and glycogen synthesis
- insulin activates insulin sensitive kinase and PP1
- dephosphorylate glycogen synthase - activating
- dephosphorylate phosphorylase kinase - inactivating
phosphorylate G subunit of PP1 at a different site, activating the phosphatase to enter glycogen particles
allosteric regulation of liver phosphorylase
how does it differ from regulation of muscle phosphorylase
- no large variation in AMP so AMP doesn’t activate the b form
- level of a form is regulated by glucose binding
- phosphorylase a is a glucose sensor in the liver, binding of glucose to phosphorylase converts it to an inactive T form, promotes dephosphorylation by PP1 (inhibit further production of glucose by liver glycogenolysis when BGC is high)
- also activate glycogen synthesis as phosphorylase a->b leads to PP1 release, which binds tightly to a form, PP1 can then activate glycogen synthase
((there is much more phosphorylase than PP1 so most phosphorylase has to be converted to the bform to release sufficient PP1 to increase glycogen synthase activity))
AMP dependent protein kinase
allosteric activation by AMP. phosphorylation of AMPK by and upstream kinase is needed for this.
AMPK limits further utilisation of ATP by inhibiting glycogen, FA and cholesterol synthesis.
advantages of protein phosphorylation for metabolic control
- signal ampl as enzymes have multiple targets
- coordination between regulatory networks
- increase sensitivity - small change in initial signal causes large change in end product (small change in relative kinase and phosphatase Vmaxes causes larger change in proportion of phosphorylated substrate with enzymes close to saturation (‘switch’ - more binary response) … is enzymes not near saturation, decrease in protein conc decreases kinase activity, slower change
- regulation of kinase and phosphatase in opposite directions gives greater sensitivity
purpose of PP1
removes all the phosphates involved in the regulation of glycogen metabolism, decreases rate of glycogen breakdown and accelerates glycogen synthesis
AMP
a sensitive indicator of cellular energy levels via the adenylate kinase equilibrium
what enhances adenylate cyclase activity in the liver
glucagon
B-adrenergic agonists
increases cAMP, activate PKA amd phosphorylate 6PF2K/F26BPase
what is 6PF2K/F26BPase?
a bifunctional enzyme found in the liver which is phosphorylated by PKA, this inhibits the kinase activity and stimulates the phosphatase activity so get more F6P and less F26BP
F26BP and liver glycolysis/ gluconeogenesis
allosteric activator of PFK1 and inhibitor of F16BPase
causes enhanced glycolysis and inhibition of gluconeogenesis
pyruvate kinase - allostery
covalent mod
inhibited by ATP and alanine, activiated by F16BP (feedforward)
inhibited by phosphorylation prior to saturation
Ca2+ linked hormones and gluconeogenesis
Ca2+-CaM dependent protein kinase phosphorylates pyruvate kinase, inhibiting it.
so promoting gluconeogenesis
why does elevated cAMP activate glycolysis in muscle but inhibit glycolysis in liver?
in the liver, 6PF2K/F26BPase and PK are phosphorylated and inhibited, whereas the musce isoforms lack a PKA phosphorylation site
PEPCK acetylation
enhanced by high glucose, decreased by high amino acid. promotes degradation of the protein
regulation of glucokinase by a regulatory protein
not inhibited by G6P but is inhibited by a regulatory protein. F6P binds the regulatory protein and reinforces the inhibition
the inhibition is antagonised by F1P - ensures liver takes up both glucose and fructose following a meal.(?)
glucose transport
GLUT1,3 = present in all cells, low Km GLUT2 = liver, pancreatic B cells, high Km so uptake dep on BGC GLUT4 = muscle, adipose, recruited to plasma membrane by insulin
control through changes in enzyme concentration
longer term, change gene expression
control through changes in enzyme concentration - liver glycolysis and gluconeogenesis
insulin - stimulates expr of PFK1, PK, 6PF2K/F26BPase
glucagon - inhibits expression of these enzymes and stimulates expression of PEPCK and F16BPase
CRE
cAMP response element
palindromic DNA sequence, mediates effects of cAMP on transcription
CREB
cAMP response element binding protein
dimerises, binds to CRE
phosphorylation of CREB by PKA promotes dimerisation and enhances transcriptional activation
PGC-1
transcriptional coactivator upon which hormonal control of liver glucose metabolism converges,
induced in liver by fasting and upregulates PEPCK, G6Pase, F16BPase
glucose-responsive TF in the liver
ChREBP=a TF which binds ChRE in the PK promoter. DNA binding is inhibited by PKA and AMPK
metabolic response to hypoxia
induction of the TF HIF1 which increases expression of glycolytic enzymes
2 ways HIF functions as a TF
- oxygen promotes HIF degradation: a prolyl-4-hydroxylase uses oxygen to hydroxylate HIF1a. this promotes recognition by the VHL tumour suppressor protein, part of a ubiquitin ligase complex so pomoting HIF1a proteolysis
Hydroxylation at an asparagine residue blocks interaction with the CTD of p300/CBP coactivators
tumour metabolism
Wahrburg effect - tumours have high rates of aerobic glycolysis, even in the presence of oxygen
p53 and glycolysis in normal cells
activation of p53 inhibits glycolysis by activating TIGAR
TIGAR has F26BPase activity - inhibiting glycolytic flux instead diverts flux into the pentose phosphate pathway, can produce NADPH and protect against oxidative stress
pyruvate kinase in tumour cells
isoform called PKM2 is expressed
phosphotyrosine motifs produced by GF signalling bind PKM2, releasing F16BP, an allosteric activator. this causes build-up of upstream intermediates and diversion of glucose into lipid synthesis
PGAM1 in tumour cells
phosphoglycerate mutase 1 - expr neg reg by p53, controls levels of 3- and 2- phosphoglycerate. increases flux in the PPP and serine biosynthesis
HIF1 in tumour cells
active as tumours often have chronic hypoxia. HIF1 stabilises p53, turns on glycolysis and off flux in the TCA cycle
= by inducing PDK1, a protein kinase which phosphorylates and inactivates PDH
PKM2 in cell’s response to oxidative stress
enzyme oxidised upon oxidative stress, decreasing its activity, which diverts flux into the PPP
link between regulation of cell cycle and cell metabolism
D cyclins and their CDKs are active in G1 and needed for cell division. phosphorylation of PFK1 and PKM2 by cyclinD3-CDK6 inhibits these enzymes, increasing flow of intermediates into the PPP and serine biosynthesis pathways as needed for cell growth/ division
cryo electron tomography
collect multiple projection images of a thick, vitrified specimen, construct 3D images
resolution limit 4-5 nm so can only observe large protein complexes - but if a macromolecule’s structure is available, can use pattern recognition techniques to identify
mechanisms of compartmentation
sequetration within cellular organelles
compartmentation by binding
problem with breaking open cells and measuring metabolite concentrations
are measuring total concentration, which may be much greater than free conc in the cytosol
compartmentation by binding
muscles have low free cytosolic ADP due to binding of ADP to actin
how does a metabolite gradient form firefly luciferase + ATP
firefly luciferase shows ATP-dependent luminescence so can be used as a probe for intracellular ATP concentration
elevated glucose concentrations and pancreatic B cells
- increased glucose leads to increased glycolysis and TCA flux so elevated ATP.
- closure of ATP-sensitive K+ channels, PM depolarisation, Ca2+ influx through VG Ca2+ channels and insulin secretion by Ca2+ mediated exocytosis
changes in PM and mitochondrial ATP were closely matched, suggesting mito are close to PM and provide a localised pool of ATP. mito may also have special access to Ca2+, which activates TCA.
protein-protein interactions
protein concentrations in vivo are very high - may enable protein-protein interactions which are not possible in dilute cell extracts
substrate channeling
enzyme complex formation - substrates are passed between enzyme active sites without fully equilibrating with the bulk phase.
advantages of substrate channeling
- high flux with low int conc
- isolate ints from competing rxns
- protect unstable intermediates
- faster response
- regulation of flux
example of substrate channeling - hexokinase
brain HK is associated with mito
skeletal muscle associates with mito in an insulin dependent fashion - HK binds porinwhich forms a pore in the membrane through which metabolites can pass. HK has preferential access to ATP generated within the mitochondria - this coordinates the initial step of glycolysis with mito OXPHOS. (shown by supplying mito with 32P and unlabelled ATP)
creatine phosphate shuttle
- in skeletal muscle, there are creatine kinase isoforms associated with the myofibril M band ATPase and inner mitochondrial membrane
- mitochondrial isoform generates PCr at the mitochondria, preferentially using mitochondrial-derived ATP.
- The PCr diffuses to the myofibrils and is used to phosphorylate ADP to ATP by the cytosolic creatine kinase
- creatine diffuses back to the mitochondria to be reused
- shuttle is effective as though there are diffusion gradients for ATP to the myofibril and ADP to the mitochondria, their concentrations are low: Cr and PCr make more effective transport molecules as they have higher concentrations
- this is how netATP transport occurs.
GPDH1 in drosophila flight muscle
- GPDH1 is an isoform of glycerol 3 phosphate DH localised to the Z discs and M lines of drosophila flight muscle. results in colocalisation of aldolase and GAPDH
- this localisation is due to a tripeptide at the CTD of GPDH1 - expression of a different isoform lacking this tripeptide does not localsie. aldolase/ GAPDH also don’t localise - flies couldn’t fly despite having almost normal levels of these enzymes
PI3K regulation of glycolysis through mobilisation of aldolase
GFs/ insulin stimulate activation of Rac via PI3K. leads to distruption of actin cytoskeleton, release of alsolase which is bound to filamentous actin
increases aldolase activity and glycolytic lfux
glycogen synthase and glucokinase mobilisation
- resting liver - glycogen synthase not localised
- presence of glucose - glycogen synthase migrates to the periphery. glucokinase colocalises
- glycogen first laid down at periphery
where do metabolic pathways come from?
pathways are evolutionarily ancient and happen slowly non-enzymatically,eg in conditions mimicking the archaean ocean environment
topology of metabolic networks
scale free networks linked together by a few highly connected substrates (coenzymes)
features of scale free networks
- probability that a given substrate participates in k rxns follows a power law distribution (rather than a poisson distribution for a random network)
- any 2 nodes are connected by relatively short paths (enzymatic reactions), average is 3 (indep of network size)
measuring interconnectivity of a scale free network
network diameter - shortest biochemical pathway averaged over all pairs of substrates - remarkably small
elementary flux modes
break-down of metabolic pathway, the smallest sub-networks which enable the metabolic system to operate at steady state
network robustness
efficiency
related to number of elementary modes
(resistance to effect of mutation)
but increasing number of elementary modes comes at an energetic cost, ie must synthesise more enzymes - need balance
mainly get network redundancy through gene duplication, have isoenzymes where high flux is needed
which enzymes of a pathway have potential to control flux?
enzymes which catalyse a reaction whose rate is comparable to pathway flux, far from equilibrium
mass action ratio
[products]/[substrates]
methods to identify flux-controlling enzymes in a metabolic pathway
- enzymes with a low Vmax - assume these are rate-limitin. however this suggests aldolase and enolase for heart muscle glycolysis too
- identify enzymes catalysig reactions far from equilibrium (mass action ratios much less than the equilibrium constants) - suggests usual 3
- search for cross-over points - when pathway flux is stimulated, there will be a decrease in concentration of intermediates prior to the controlling enzyme and increase in concentration of intermediates after.
- look for control points at start of pathways/ immediately after branchpoints - this is a good place for control as it prevents accumulation of later intermediates. eg PFK1 catalyses the first committed step in glycolysis
how can we quantify the degree of control exerted by each enzyme in a pathway?
metabolic control analysis
metabolic control analysis
links properties of a metabolic pathway (eg flux, metabolite concs) to activities of component enzymes
flux control coefficient
fractional change in pathway flux due to a fractional change in enzyme concentration
sum of the flux control coefficients for all enzymes in a linear pathway is 1
shows the level of control an enzyme has over the total flux, this changes with different flux levels
flux control is normally distributed throughout all enzymes in the pathway, there is not one truly rate-limiting enzyme
true rate-limiting enzyme
flux control coefficient = 1
but for most systems, flux control s distributed throughout the pathway
determining FCCs of enzymes/ transporters of mitochondrial OXPHOS
titrate their activities with inhibitors
flux control shown to be distributed, this distribution depends on rate of respiration
studying flux in the TCA cycle - citrate synthase
disrupt chromosomal gene, transform cells with a plasmid with CS gene and tac promoter (trp+lac) - vary the concentration of IPTG to alter levels of CS expression
cycle flux close to 0 when incubated with glucose and high/ equ when incubated with acetate.
(FCC=slope of line, detect in WT steady state)
why use Metabolic Control Analysis?
- test whether an enzyme thought to be important in vitro is really important in a cell
- dispels simple notions eg that a non-equilibrium enzyme is one with a high FCC
- applications in biotech - metabolic pathway engineering
- help choose drug targets
tryptophan biosynthesis - pathway engineering
transforming cells with plasmids overexpressing 1/5 enzymes does not affect rate of tryptophan biosynthesis, however transforming with a plasmid which expresses all 5 enzymes does
metabolome
metabolite complement of a cell/ tissue
changing concentration of an enzyme …
little effect on pathway flux
large effect on metabolite concentrations, sum to 0 - more easily detected
fluorescence
emission of radiation when a molecule in an excited state returns to its ground state, normally appears at a longer WL than the incident light as some vibrational energy is lost to the surroundings
why is fluorescence so sensitive
no background from the excitiation source as detection wl is different
types of modern fluorescence microscopy
wide field, confocal, multi-photon, light sheet
confocal fluorescence microscopy vs wide field
wide field is low cose, low photobleaching but confocal has better depth resolution, producing images as defined stacks which can be compiled into a 3D image
multi-photon fluorescence microscopy
better depth resolution than confocal
… if 2 long WL photons hit a fluorophore at the same time, the energy is combined
longer WL laser gives better penetration through tissue + causes less damage to specimen
light sheet fluorescence microscopy
even better depth resolution
laser light source orthogonal to objective, only illuminate a very thin sheet of the tissue in same plane as imaging objective’s focal plane
- optical sectioning reduces background so gives high-contrast image. reduces photodamage/ stress on sample, can look at large specimen
3 types of super-resolution microscopy
SIM: structured illumination
STED: stimulated emission-depletion
PALM/ STORM single molecule localisation
SIM: structured illumination
sample is excited by laser passing light through optical grating, non-uniform illumination, creating striped interference pattern,moire fringe. use info from structures below the diffraction limit to generate coded images which must be combined mathematically.
STED: stimulated emission-depletion
first laser excites fluorophore,
donut shaped depleting laser excites electrons again so when they return to ground state a different fluorescence wavelength, which s not detected, is emitted. so detect emission from a smaller area of sample.
PALM/ STORM single molecule localisation
use photoactivatable/ photoswitchable dyes/ proteins to limit number of simultaneously emitting particles
- molecules are detected as diffraction-limited spots as gaussian distribution - know object must be in centre of this
- use single molecule positions from 1000s of images, eah with different emitters, to build up density map
pH measurement
fluorescence of dye BCECF at 500nm is brighter at higher pHs
this alone would be unreliable as fluorescence also depends on optical path length
to control for this, also measure fluorescence at 450nm (fluorescence indep of pH here) and calculate ratio 500nm/450nm.
Ca2+ measurement
fura-2 enters cell in form with esterified carboxyl groups. ester groups cleaved off internally, probe binds Ca2+ with high affinity and selectivity. binding of Ca2+ shifts fluorescence excitation spectrum to shorter WLs, measure ration 350nm:385nm
measuring intracellular cAMP
use derivative of PKA: regulatory and catalytic subunits both tagged with fluorophore. low cAMP: subunits associate, FRET occurs. cAMP: subunits dissociate, no FRET so emission at different WL
FRET: ATP biosensor
efficiency of FRET increases in presence of ATP as E subunit of ATP synthase retracts, drawing the 2 fluorophores closer together
fluorescence polarisation
there is a delay between the absorption and emission of light by a fluorophore, during which it can rotate- use to determine tumbling speed and intracellular viscosity
intracellular viscosity/ translational diffusion
intracellular viscosity is low, as shown by fluorescence polarisation with small molecules
however translational diffusion of larger molecules is significantly hindered
NMR: physical principles
nuclei with non-zero spin have a magnetic moment and in the presence of an applied magnetic field, assume discrete energy levels. the magnetic moments of the nuclei process around the applied field . an oscillating magnetic field is applied perpendicular to the static field, which induces transitions of nuclei between the two energy levels, which is detected by the NMR spectrophotometer
???
NMR chemical shift
nuclei of one element resonate over a range of different frequencies depending on chemical environment
express in ppm to normalise
NMR intensity
area under a peak is proportional to number of nuclei giving rise to it
NMR peak width
inversely proportional to spin-spin relaxation time eg which depends on tumbling rate of the nucleus. smaller T2 for a larger molecule so larger line width
31-P NMR and bioenergetics
eg during exercise, in muscle can see decrease in PCr, shift in Pi as pH decreases, constant ATP concentration
rate of PCr resynthesis after exercise = measure of muscle oxidative capacity
???
NMR imaging
MRI can give anatomical information through the mapping distribution of water molecules
magnetic field gradient is applied across sample, resonance frequency of water protons varies depending on their spatial position within the gradient.
functional MRI
MRI can give anatomical information through the mapping distribution of water molecules
- frequency at which water resonates depends on their position in the magnetic field gradient
- increased brain activity increases glucose use and blood flow to that part of the brain more than oxygen consumption is increased - so oxygen level increases, deoxyHb decreases.
- deoxyHb is paramagnetic so influences the relaxation of nearby water molecules
- decreased deoxyHb increases MRI signal intensity in nearby brain areas
greater dynamic response???
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