Control of metabolism Flashcards
(36 cards)
Carb metabolism in skeletal muscle and liver
Glucose powers muscle contraction: glycolysis supplies myosin ATPase. Though ATP pool buffered by phosphocreatine during intense exercise, PCr limited so glycogen quickly mobilised-> glucose-6-Pi . Anaerobic glycolysis-> lactate+ H+ to blood, hence only short periods of intense activity possible.
Prolonged exercise: ATP produced more slowly from aerobic oxidation of glucose-> exercise less intense.
The liver buffers blood glucose in response to glucose conc and hormonal signals, by glycogenolysis or gluconeogenesis (only possible here). The steps catalysed by hexokinase, PFK1 and pyruvate kinase are not reversible, unless enzymatically in the gluconeogenic pathway by pyruvate carboxylase+ phosphoenolpyruvate carboxykinase, F1,6B and glucose 6-phosphatase respectively. Lactate and alanine released into blood by muscle can be converted to glucose in liver (via gluconeogenic pathway) in the Cori cycle.
Control of enzyme activity at substrate level
1)Substrate level ctrl: rate most sensitive to changes in [S] at/ below Km, where response is proportional (double [S]-> max. double rate). Change in [S] rarely act as signals for regulation of flux-ctrling enzymes. Exception: regulation of glucokinase/hexokinase IV in pancreatic beta cells+ liver; Hexokinase isoform has high Km for glucose comparable to blood conc. In pancreatic beta cells (secrete insulin, response to increase in [glucose]), glucokinase was hypothesised to be “glucose sensor”- increase in blood glucose thought to increase glucokinase activity-> increased glycolytic flux in beta-cell and insulin secretion. Supported by transgenic mice (exp yeast hexokinase in beta cells increased insulin secretion at low [glucose]- yeast enzyme has lower glucose Km, active at low plasma concs. Transgenic mice also less susceptible to diabetes). NB both liver+ beta cell membranes freely permeable to glucose due to high levels of insulin-independent GLUT2s, so cytoplasmic conc roughly= blood conc.
Flux vs rate
Flux: system property. Describes overall rate of multi-component system (e.g., glycolytic pathway).
Rate: local property. Describes rate of single enzyme.
Insect flight muscle flux increases x100 when flight starts w/ very little change in [ATP].
Enzyme activity control through cooperativity
2) Cooperativity: increases response of enzyme to change in substrate conc. Bind 1st substrate molecule-> affect subsequent binding. Increase affinity= +ve cooperativity+ vice versa. 2 models:
* Monod-Wyman-Changeux (MWC)- concerted model. Binding 1st molecule moves equilibrium toward relaxed form, facilitating further binding. Can oonly explain +ve cooperativity
* Koshland-Nemethy-Filmer (KNF)- sequential model. 1st binding-> conformational change of bound subunit from T to R form-> next subunit changes T to R. Can explain +ve and -ve cooperativity.
Sigmoidal substrate binding curve. For protein P w/ n binding sites for ligand L w/large increase in affinity following 1st L binding, P only exists in free or fully bound PLn form. When n=1, curve like Michaelis Menten, then gets increasingly sharply sigmoidal as n increases (ultrasensitivity).
Analysing curves of real enzymes, calculated n usually=underestimate+ not integer bc assumption that infinite degree of cooperativity not valid. Calculated n=Hill coefficient- measure of cooperativity. Multiple subunits not prerequisite for coop, e.g., glucokinase monomeric, but sigmoidal saturation curve because conformations interconvert slowly. Low affinity (E’) form predominates at low [S], high affinity form E when high [S]+ reverts back slowly after binding+ unbinding-> enhanced sensitivity to [S].
Enzyme control by allostery
3) Allostery: Regulatory enzymes often show coop wrt S binding+ ctrled by allosteric effectors (NB exception= muscle isoform of hexokinase allosterically inhibited by glucose-6-Pi but has only 1 subunit+ doesn’t show cooperativity w/rt binding its S (glucose)). Allosteric activators stabilise R state in MWC model+-> conformational change-> activation.
Muscle glycolysis: ctrled at level of glucose uptake, hexokinase, PFK1, pyruvate kinase. PFK1 allosterically inhibited by ATP, inhibition relived by AMP. Exercise-> large increase [AMP] due to near-equilibrium reaction catalysed by adenulate cyclase (2adp-> amp+atp)-> AMP= v sensitive energy status indicator (15% [ATP] decrease-> 3 fold [AMP] increase). Increase PFK1 activity-> increase steady state [F-1,6-BP] which allosterically activates pyruvate kinase and decrease [G-6-P] which inhibits hexokinase-> coordinated increase in activity of 3 enzymes controlling flux in glycolysis
Enzyme control through substrate cycles
4) Substrate cycles: PFK1+ Fru-1,6-BPase catalyse substrate cycle w/ possibility for ATP hydrolysis in absence of net flux through pathway. Cycle can amplify regulatory response, e.g., AMP activate PFK1, inhibit Fru-1,6-BPase. 2-fold change in activity from PFK1 flux 100 and Fru-1,6-BPase flux 98 in both enzymes raises net glycolysis flux 2-> 151. Signal amplification paid for by ATP hydrolysis, free energy released as heat (also good for keeping muscles warm). Perpetually active muscles (e.g., heart) don’t have extreme energy demand variations, lack F-1,6-BPase.
Enzyme control through covalent modification
5) Covalent modification: Pi. Most phosphorylated proteins Pi’d on Ser/Thr, 1% on Tyr, but 428 ser/thr kinases and 90 Tyr kinases, so each ser/thr kinase acts on many more distinct residues than Tyr kinase. Only 147 protein phosphatases, only 40 act on Piser/thr, so have many targets, and many have distinct regulatory subunits for direction to specific sites. Most tyr phosphatases have regulatory domains within the same polypeptides. 5-10% genes involved in regulating protein Pi events, e.g., glycogen breakdown+ synth.
Lys acetylation- widespread acetylation/metabolic enzymes suggests importance in ctrl/ cell f(x). mass spec shows almost all enzymes in glycolysis, gluconeogenesis, TCA+ urea cycles, fatty acid/glycogen metabolism acetylated in liver+ there are changes in profile of acetylated metabolic enzymes w/ carbon source. Lys acetylation by acetyl CoA as donor- model in which changes in cellular pools of acetyl CoA regulate acetylation+ activity of metabolic enzymes.
Muscle glycogen breakdown and synthesis: Glycogen phosphorylase
Glycogen phosphorylase: subject to allosteric ctrl+ Pi. Less active b form-> pi Ser14/both subunits-> active a form. Pi+ AMP allosterically activate, G-6-P+ ATP inhibit. If b form already fully activated allosterically, further Pi has little further effect. Regulation can be viewed in terms of concerted/MWC model.
Crystal structures of phosphorylase b show 4 forms: T state protomer-> R state protomer (N-terminal region swings to form new interactions), T state dimer and R state dimer (new inter-subunit interactions rotate dimer interface 10o, opening binding sites for Po and UPDG).
Muscle glycogen breakdown and synthesis: phosphorylase kinase
Phosphorylase Pi’d by phosphorylase kinase, which is Pi’d by PKA (cAMP-dependent). So glycogen breakdown/ synthesis co-ordinately regulated by cascade of protein Pis. Adrenaline-> activate adenylate cyclase-> atp to cAMP-> activate PKA by promoting dissociation of regulatory and catalytic subunits.
Phosphorylase kinase- large complex with composition (alpha, beta, gamma, delta)4. Fully activated by PKA Pi’ing alpha and beta subunits. Non-Pi’d form can be partly activated by Ca2+ binding delta, which is calmodulin- important in muscle (contraction triggered by Ca release). In live, Ca2+ release produced by hormone activation of phosphoinositide cascade
Muscle glycogen breakdown and synthesis: Glycogen synthase
Glycogen synthase: Pi’d on any of 7 Ser by 5+ protein kinases. Pi inhibits activity by increasing Km for UDP-glucose substrate and Kd (dissociation constant) for activator G-6-P, while Kds for inhibitors ATP+Pi reduced- activity changes gradually as Pi states of each site changes. Pi of phosphorylase+ glycogen synthase-> glycogen synth and degradation coordinated w/ muscle contraction. Increased [Ca] following neural stimulation/ increased cAMP following adrenaline stimulation-> rapid activation phosphorylase+ deactivation/glycogen synthase (by Pi site 2 by PKA/ phosphorylase kinase)
Muscle glycogen breakdown and synthesis: Protein phosphatase
Protein phosphatase 1 (PP1)-> remove all Pi groups in regulation of metabolism. Activity decreases glycogen breakdown+ accelerates glycogen synth. Catalytic subunit of PP1 alone ineffective (low affinity for glycogen). High affinity by association w/ ‘glycogen binding’ G subunit. Pi of G by PKA prevents it binding catalytic subunit/ PP1, inactivating it. PP1 also has inhibitor which acts when Pi’d by PKA. Thus, glycogen degradation switches on by cAMP+ Pi of inhibitor-1+ G subunits inhibits PP1, keeps phosphorylase active+ glycogen synthase off.
Insulin activates PP1. Enzyme Pi’s G subunit at different state to that modified by PKA, activating it. Consequent de-Pi of phosphorylase kinase, phosphorylase, glycogen synthase-> glycogen synth, block degradation.
Liver glycogen breakdown and synthesis: overview
AMP does NOT activate b form phosphorylase, a form regulated by glucose binding, acts as glucose sensor. Binding glucose shifts equilibrium to T form, promotes de-Pi of Ser14 by PP1-? B form inactivated. When bloof [glucose] high, glycogenolysis inhibited. High [glucose] activate glucose synth: PP1-bound a form to b conversion-> PP1 release, free to activate glycogen synthase.
~10a:1phosphatase, so glycogen synthase activity only increases if most of a converted to b. recent work suggests this can be overridden by increase in G6-P-> promote de-Pi+ activation of glycogen synthase.
Liver glycogen breakdown and synthesis: AMPK, and advantages of Pi for enzyme ctrl
5’-AMP ctrls AMPK activity: initially though to only ctrl HMG-CoA reductase (cholesterol synth)+ acetyl CoA-carboxylase (fatty acid synth), later found multiple cellular targets of AMPK (e.g., blocking lipolysis in adipose, promoting fatty acid oxidation in muscle). Allosteric activation of AMPK: binding AMP-> AMPK extrudes lipid anchor that tethers plasma membrane close to LKB1 (upstream kinase)-> LKB1 Pi’s Thr172-> activation of AMPK.
Advantages of Pi for enzyme ctrl:
* Signal amplification, e.g., cAMP (conc 1uM) activates phosphorylase (~100uM)
* Co-ordination between different regulatory networks
* Increased sensitivity (small change-> large response) compared to ligand binding (cooperativity increases sensitivity, but max change still=[ligand]n). covalent modification (Pi)-> zero-order ultrasensitivity possible- small change in kinase/phosphatase Vmax-> large change in proportion of Pi substrate when both enzymes close to saturation (much less sensitive if not saturated)- if kinase+ phosphatase regulated in opposite directions, even greater sensitivity (e.g., PKA pi’s+ activates phosphorylase kinase, inhibits PP1 by Pi’ing G and inhibitor-1)
Control of liver glycolysis and gluconeogenesis: agonists, PK, CaM
Glucagon+ beta-adrenergic agonists enhance adenylate cyclase activity-> increase [cAMP]-> activate PKA, Pi pyruvate kinase+ (6PF-2-K/Fru-2,6-P2ase) bifunctional enzyme at Ser- Pi of the bifunctional enzyme-> inhibition of its kinase+ activation of its phosphatase-> [F-2,6-VP] falls (allosteric activator of PFK1+ inhibitor of Fru-1,6-P2ase-> increase gluconeogenesis, inhibition/glycolysis, decrease in F-1,6-BP. So increase PK switches glycolysis off.
PK allosterically inhibited by ATP+ alanine; activated by F-1,6-BP; increase Km for phosphoenolpyruvate by Pi (decrease activation by F-1,6-BP+ enhancing Ala/ATP inhibition). In saturating concs of F-1,6-BP, pi has no effect. Enzyme is better PKA substrate when allosterically inhibited.
Ca2+-calmodulin- dependent protein kinase catalysed Pi of pyruvate kinase @both Ser+Thr-> smaller effect of Ca2+ linked hormones.
Control of luver glycolysis and gluconeogenesis: insulin, cAMP, Ischaemia, PEPCK acetylation and glukokinase
Insulin enhances low Km phosphodiesterase activity, lowering intracellular cAMP (inverse to above effect).
High cAMP activates glycolysis in skeletal muscle but inhibit it+ activate gluconeogenesis in liver: liver isoforms of PK+ 6PF-2-K/Fru-2,6-P2ase Pi’d+ inhibited while muscle isoforms lack PKA Pi sites.
Normal heart muscle- adrenaline-mediated Pi of 6PF-2-K/Fru-2,6-P2ase -> increase Fru-2,6-P2, faster glycolysis. Ischaemic muscle- Pi of 6PF-2-K/Fru-2,6-P2ase by AMP-dependent protein kinase increases kinase activity, stimulating glycolysis.
PEPCK acetylation enhanced by high glucose, decreased by increased [aa]. Acetylation->protein degradation.
Liver glucokinase (not inhibited bu G6-P like muscle hexokinase) inhibited by regulatory protein, reinforced by F6P binding reg. protein, preventing futile cycle between G+ G6P. Inhibition antagonised by F1P, stimulating glucokinase- ensures both glucose+ fructose taken up by liver after mean
Glucose transport
GLUT1+3- nearly all mammalian cells, basal transport (low Km ~1mM, much less than normal blood level)
GLUT2- liver, beta cells, high Km ~15-20mM- uptake rate proportional to blood level.
GLUT4 Km ~5mM, recruited to plasma mem of muscle+ fat cells by insulin to promote uptake.
GLUTs stored in vesicles-> insulin/receptor binding-> vesicles fuse membrane-> when insulin drops transporters removed by endocytosis-> smaller vesicles fuse larger endosome-> patches of GLUT enriched endosome bud off to vesicles for next use. Image GLUT4-GFP in real time in transgenic mice-> response ~30s.
Enzymatic control though enzyme concentration changes: Hormones, CREs, hypoxia response
Longer term (several hours)
Rise in insulin-> expression of PFK1, pyruvate kinase+ 6PF-2-K/Fru-2,6-P2ase in liver. Glucagon inhibits their exp, stimulates exp PEPCK+ Fru-1,6-BPase. PEPCK gene first isolated for an enzyme involved in hepatic gluconeogenesis- 500bp region of promoter has reg seqs mediating actions of hormones: IRE (insulin response element), GRE (glucocorticoid), TRE (thyroid hormone), CREI+CREII (cAMP)
CREs: 8bp palindromes bound by Leu zipper CREB. Dimerization via C terminal Leu zipper domain (promoted by Pi by PKA) brings together 2 basic DNA binding modules that bind CRE-> activation.
Hormonal ctrl/liver glucose metabolism by transcriptional activator PGC-1- induced by fasting-> PEPCK, G6 phosphatase+ F16Bphosphatase upregulation. Also a glucose- responsive TF: high carb diet-> transcription/ genes for glycolysis and lipid synth enzymes- ChRE binding protein (ChREBP)= basic HLH Leu zipper TF binds carb responsive element (ChRE) in promoter of L-type pyruvate kinase gene. DNA binding inhibited by Pi of PKA+ by AMPK.
Hypoxia response- normal cells-> induction of HIF-1 (basic HLH TF w/ alpha+ beta subunits)- increases exp/ glycolytic enzymes. Oxygen promotes: HIF-1alpha degradation; a prolyl-4-hydroxylase that uses oxygen as co-substrate, hydroxylates P564+P402 of HIF-1alpha- this promotes interaction of alpha w/ von Hippel-Landau tumour suppressor protein (recognition component of UQ ligase, promotes UQ-dependent HIF-1alpha proteolysis)- blocks TF activity of HIF-1 by blocking CTD HIF-1alpha interaction w/ p300/ CBP transcriptional co-activators by hydroxylating asp N803. NB beta subunit AKA ARNT.
Tumour metabolism: Warburg
Recently, molecular basis for altered metabolism elucidated-> new ways to ID+ treat tumours.
Higher rates of aerobic glycolysis: Warburg showed this in majority/ animal tumours . Hypoxic env of tumours selects cells adapted to chronic hypoxia. HIF-1 stabilised p53 (suggests p53 has direct role as TF in hypoxia). Mutant p53 stimulates transcription/ H2K by binding promoter (suggests role in tumour metabolism). Oncogene v-SRC induces HIF-1 exp-> glycolysis (+ switches off flux via TCA). HIF-1 also induces PDK1 gene-> protein kinase that Pi’s E1 subunit/ PDH, inactivating it.
P53 in normal cells- activation inhibits glycolysis by activating TIGAR (has F-2,6 Bphosphatase activity). Inhibition/ glycolytic pathway-> flux to pentose phosphate pathway-> NADPH production+ reduction of ROS.
Tumour metabolism: PKM2, PGAM1, cell cycle
PKM2 (splice isoform of PK) exp in tumour cells: PiTyr motifs produced by growth factor signalling via protein Tyr kinases bind PKM2 directly, releasing its allosteric activator F1,6BP. PKM2 inhibition-> upstream intermediates in glycolytic pathway build up, glucose diverted to lipid synth. C358 in PKM2 oxidised by H2O2, decreasing activity+ pyruvate formation (by diverting flux to pentose Pi pathway (PPP)). Increased PEP inhibits triosephosphate isomerase, increase flux to PPP.
PGAM1 exp -vely reg by p53. Reg biosynth by ctrling 3-PG+ 2-PG levels. 3PG binds/inhibits 6-phosphogluconate dehydrogenase in PPP; 2-PG activates 3- phosphoglycerate dehydrogenase-> feedback ctrl on 3-PG levels by increasing flux to Ser biosynth pathway.
Cell cycle+ metabolism: D-type cyclins CDK4+6 needed for cell divition. Peak activity early G1. Pi of PFK1+ PKM2 by cyclin D3-CDK6 complex inhibits them, promoting dissociation of tetramers-> less active dimers, directing glycolytic intermediates-> PPP+ Ser synth pathway.
Organisation of cellular metabolism
(Cryo-ET- can often be used for study)
Compartmentation of metabolites by sequestration in organelles common. Total concs from acid extracts from
cells> free conc in cytosol/ < conc in organelle. E.g., brain cytosol [ADP]«_space;total bc sequestered in mt. Free cytosolic conc determined measuring tissue conc of creatine kinase substrates.
Compartmentation by binding- e.g., low muscle [ADP] due to binding actin- many enzymes+ substrate compartmentalised this way, e.g., PK, pyruvate, F-1,6-BP.
Metabolite gradients- w.g., ATP microdomains in beta cells. Trace w/ firefly luciferase- ATP dependent (Km ~1mM) luminescence to probe intracellular [ATP], targeting it to subcellular locations. High [glucose]-> higher glycolysis+ TCA flux-> ATP-sensitive K+ channels close-> membrane depolarisation, Ca2+ influx via VG Ca2+ channels-> insulin secretion. Changes in [ATP] @ plasma membrane+ mt closely matched, suggesting mt close to mem provide local ATP pool+ may have privileged Ca2+ access, which activates TCA, creating feed-forward loop.
Protein-protein interactions- high [protein] in vivo promotes interactions (may also be enhanced by solvent exclusion effects in solutions of high conc macromolecules, can cause large increases in activity coefficients of proteins) not seen in diluted extracts.
Creatine cycle/Keq
Substrate channelling
substrates passed between diff active sites w/out fully equilibrating w/ bulk phase. Potential advantages: high flux @low intermediate concs; isolate intermediates from competing reactions; protect unstable intermediates; circumvent unfavourable equilibria; faster response, reduce lag time in transients between stead states; flux regulation. Evidence: studies on isolated enzyme systems (evidence of direct enzyme-> enzyme handover as intermediates never diffused freely/ released to solvent).
Substrate channelling: Hexokinase
Hexokinase in brain 80-90% activity in mt. Insulin sensitive tissues (e.g., skeletal muscle)- mt binding enzyme insulin-dependent. HK binds porin (outer mt mem). Isolated mt show localised HK has preferential access to intra-mt made ATP (e.g., mt incubated w/ P-32 isotope+ unlabelled ATP, G-6-P produced had specific activity= that of Pi, indicating ATP produced by ox. Pi= ATP source for HK, not unlabelled ATP from medium. Channelling of ATP thought to be means of coordinating initial step/ glucose metabolism w/ mt ox. Pi. HKII binding heart mt: perfusion of isolated hearts w/ cell-mem peptide w/ HKII mt binding motif (TAT-HKII) which displaces HKII for 15 min-> mem depol, mt swell-> structural disruption of cardiac tissue. TAT-CON ctrl peptide-> no effect.
Substrate channelling: creatine phosphate shuttle
Creatine phosphate “shuttle”: skeletal muscle creatine kinase cytosolic form assoc. w/ M band, functionally couples to myofibrillar Mg2+ ATPase. Mt CK isoform on outer surface/inner mem forms complex w/ adenine nt translocase in inner mem+ porin in outer. Isotope labelling exps show mt CK has preferential access to ATP made in mt matrix. Localisation important for:
* Acting as temporal energy buffer, maintaining [ATP, ADP, H+] during increased cellular activity.
* Maintain high local ATP/ADP ratio @ sites/rapid ATP use (e.g., myofibrils)
* Act as energy shuttle- PCr made in mt diffuses to myofibrils-> Pi ADP from myofibrillar ATPase. Creaine diffuses back to mt. Net ATP transport mt-> myofibrils via PCr diffusion- PCr/Cr better transport molecules than ATP/ADP bc present @ higher concss, esp compared to ADP.
Shuttle hypothesis has strong support from mouse strain w/ both muscle CK genes disrupted- skeletal muscles adapt w/ more+ larger mt, reducing diffusion distance between mt+ myofibrils.
