Control Of Metabolism

Front 1

Used before ATP when intensively exercising

Back 1

phosphocreatine

Front 2

3 irreversivle steps catalysed by (glycolysis)

Back 2

hexokinase

6-phospho-frukto-1-kinase (PFK1)

pyruvate kinase

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3 irreversible steps catalysed by (gluconeogenesis)

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glucose 6-phosphatase

fructose 1,6-bisphosphatase

phosphoenolpyruvate carboxykinase

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what's released by muscle and converted to what

Back 4

alanine and lactate, both go to pyruvate and glucose vio gluconeogenesis

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Control of enzyme activity

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Substrate level control

cooperativity

Allosteric effectors

Substrate cycles

Covalent modifications

Control through changes in enzyme concentration

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exception: which flux-controlling enzyme is controlled by changes in substrate concentration

Where's it found, how

Back 6

glucokinase, in beta cells and liver.

Has high Km for glucose, comparable to [glu] in plasma (5mM)

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another name for glucokinase

Back 7

hexokinase IV

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beta pancreatic cells and glucose

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glucose ↑→glucokinase activity ↑→ glycolytic flux ↑→insulin secretion

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Experiment to test for hexokinase control by glucose

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Expressed yeast hexokinase in mice (lower Km), so active even at low glu concentrations. Transgenic mice had lower glucose elvels and higher insulin concentration.

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sup with plasma membrane of beta and liver cells

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freely permeable to glucose: lots of insulin-independent GLUT 2 transporters - [glu] in and out close to equilibrium

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two cooperativity models

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Monod-Wyman-Changeux

Koshland-Nemethy-Filmer

Concerted and Sequential

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Hill coefficient

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n in % saturation with ligand

L^n/(Kd+L^n)

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for higher hill coefficients

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sigmoidal curve is ssteeper/ultrasensitive

Front 14

exception to the rule that regulatory enzymes show cooperativity wrt substrate binding and controlled by allosteric effectors

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muscle hexokinase, which is allosterically inhibited by glu6phosphate but has only one subunit and does not show cooperativity wrt substrate binding.

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what controls glycolysis

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level of glucose uptake

heokinase

pfk1

pyruvate kinase

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PFK1 allosteric inhibitor

what is it relieved by

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ATP (also substrate)

AMP

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pyruvate kinase allosteric activator

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fructos 1,6-bisphosphate

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effects of increase in PFK1 activity

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→F16BP↑ (allosteric actiator of pyruvate kinase)

→Glu6P↓ (inhibitor of hexocinase)

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Substrate cycles. elaborate

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Phosphofruktokinase and fructose 16biphosphatase.

substrates circled around using ATP

regulatory signal comes in (eg AMP activates PFK1 and inhibits F16BPase) and the throughput is much increased.

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phosphorylation sites

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serine threonine (tyrosine)

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how many ser/threonine and tyrosine in humans

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428 ser/threonine

90tyr

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covalent regulatory modifications

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phosphorylation and acetylation

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what can be acetylated

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lysine, uses acetyl CoA as donor

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glycogen synthase control (muscle)

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phosphorylation on several serines

phosph inhibits activity by increasing Km for substrate and Kd for activator

Kds for inhibitors ATP and Pi reduced

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substrate of glycogen synthase (muscle)

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UDP-glucose

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activator for glycogen synthase (muscle)

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glucose-6-phosphate

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metabolism-related phosphatase (muscle)

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protein phosphatase 1

responsible for the removal of all phosphates involved in the regulation of glycogen metabolism

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what does PP1 do (muscle)

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decreases the rate of glycogen breakdown and accelerates glycogen synthesis

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catalytic sub of pp1(muscle)

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low affinity for glycogen particles - ineffective

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glycogen particles have (muscle)

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phosphorylase and glycogen synthase

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what makes PP1 go to glycogen particle (muscle)

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association with glycogen-binding/G subunit

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Phosphorylation of G subunit? (muscle)

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By PKA

Prevents binding to catal subunit of PP1

Inactivates PP1

By Insulin-Sensitive Protein kinase

at different site

activates PP1

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inhibition of PP1 (muscle)

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inhibitor

when phosphorylated by PKA

inhibits phosphatase

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PP1 activator (muscle)

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insulin

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ho regulation of liver phosphorylase differs from muscle

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liver: AMP does not activate the b form and the level of a form is regulated by glucose binding.

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glucose sensor in liver

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phosphorylas a

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production of glucose

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glycogenolysis

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how many phosphorylase a per phosphatase

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10

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Advantages of protein phosphorylation for control of enzyme activity

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Signal amplification

coordination

increased sensitivity

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effect of glucagon and beta-adrenergic agonists in liver

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adenylate cyclase*→cAMP↑→PKA*→phosphorylation of:

pyruvate kinase

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase at serine residues

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6PF-2-K/Fru-2,6-P2ase does what? (liver)

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inhibits the kinase and activates phosphatase

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consequence of activation of 6PF-2-K/Fru-2,6-P2ase (liver)

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fructose-2,6-bisphosphate concentration falls

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activator and inhibitor of what is fructose 2,6-bisphosphate (liver)

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activator of PFK1

inhibitor fru-1,6-P2ase

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result of activation of PFK1 and inhibition of fru-1,6-P2ase (liver)

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enhanced glycolysis, inhibited gluconeogenesis and increase of fructose 1,6 bisphosphate

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pyruvate kinase allosteric regulators (liver)

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inhibitors ATP and alanine

activator fructose 16 bisphosphate

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phosphorylation of pyruvate kinase (liver)

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inhibits it by

increasing Km for phosphoenolpyruvate

decreasing activation by fructose-16 bisphosphate

enhancing inhibition by ATP and alanine

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effect on Ca-linked hormones on gluconeogenesis is mediated by (liver)

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Ca-calmodulin-dependent PK catalysed phosphorylation of pyruvate kinase at serine and threonine

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how insulin reverses effects of stimulatory hormones on gluconeogenesis (liver)

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by enhancing low Km phosphodiesterase activity

and

lowering cAMP levels

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how cAMP activate glycolysis in muscle and inliver oppositely inhibits glycolysis and activates gluconeogenesis

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Muscle isoform of 6PF-2-K/Fru-2,6-P2ase and pyruvate kinase lack PKA phosphorylation site

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Response to adrenaline in heart wrt Fru-2,6-P2

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adrenaline - Phosphorylation of 6PF-2-K/Fru-2,6-P2ase - rise in Fru-2,6-P2ase (liver: decrase in levels) - accelerate glycolysis

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PEPCK?

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Phosphoenolpyruvate carboxylatekinase

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PEPCK acetylation? (liver)

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enhanced by glucose

decreased by amino acids

promotes degradation

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Glucokinase regulation (liver)

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inhibited by protein binding

NOT inhibited by G6P

Fru6P binds regulatory P, reinforces inhibition, prevents futile cycle between glucose ang G6P

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activates glucokinase (liver)

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fructose 1 phosphate

ensures Glucose and Fructose uptake

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Glut 1

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nearly all mammalian cells, basal glu transport

Km =1mM

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Glut 2

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liver and pancreatic beta cells

high Km for glu (15-20 mM)

glu uptake rate proportional to blood glucose level

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Glut 3

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nearly all mammalian cells, basal glu transport Km =1mM

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Glut4

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Km 5mM

recruited to the plasma membrane of muscle and fat cells by insulin promoting glucose uptake by these cells

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Insulin stimulates expression of

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PFK1, PK and 6PF-2-K/Fru-2,6-P2ase

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Glucagon vs expression

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inhibits PFK1, PK, 6PF-2-K/Fru-2,6-P2ase

stimulates PEPCK and Fru-1,6-P2ase

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how long it takes to change enzyme concentration

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several hours

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what mediates the effect of cAMP on transcription (liver)

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CRE (cAMP response elements)

8bp palindromes

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what binds to CRE (liver)

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(cAMP response elements)

CREB (cAMP response element binding protein)

Leucine zipper

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How does CREB work(liver)

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(cAMP response element binding protein)

dimerises through C-terminal leucine zipper

brings together two basic DNA binding molecules that bind CRE

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What promotes dimerisation o CREB (liver)

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PKA - also enhances transcriptional activation

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Hormonal control of liver glucose metabolism

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transcriptional co-activator PGC-1

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What does PGC-1 do

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induced in liver by fasting

coordinate upregulation of PEPCK, G6Pase, Fru-1,6-P2ase gene expression

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transcription factor regulating Glu metabolism in liver

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Glucose-responsive transcription factor,

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ChREBP

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Carbohydrate response element binding protein

helix-loop-helix leucine zipper,

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What ChREBP binds

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ChRE in L-type pyruvate kinase promoter

DNA binding inhibited by phosphorylation by PKA and by AMPK

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Describe HIF-1

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helix-loop-helix factor from alpha and beta subunits

increases expression of genes encoding glycolytic enzymes

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effect of oxygen on HIF-1alpha

interacts with what tumor suppressor protein

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•Prolyl-4-hydroxylase +O2 hydroxylates two prolines on the HIF-1α

•Promoted interaction with von Hippel-Landau tumor supressor protein

•Ubiquitin ligase recognises Hippel-Landau P

•DESTRUCTION

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effect of oxygen on HIF-1α

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•Hydroxylation of aspargine

•blocks interaction of C terminal domain with

•p300/CBP transcriptional co-activators

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Tumors do what glycolysis

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aerobic glycolysis

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HIF-1 stabilises what and so?

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p53 → plays direct role as transcription factor in hypoxia

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mutant p53 stimulates transcription of what promoter

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HK2 (hexokinase)→ p53 has role in tumour metaobolism

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what induces HIF-1 expression

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v-SRC

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what does HIF-1 do

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turns on glycolysis and turns off flux to TCA cycle

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how HIF-1 cuts off stuff from TCA

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induces PDK1 (protein kinase)

no Pion of E1 of PDH (pyruvate dehydrogenase)

inactivated PDH

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how p53 inhibits glycolysis

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activates TIGAR

de-Pases fru-2,6-bisphosphate

inhibits glycolysis

diverts flux into pentose phosphate pathway

NADPH production

which reduses ROS (reactive oxygen species)

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TIGAR

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TP53-induced glycolysis and apoptosis regulator

has fru-2,6-bisphosphate activity

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tumors express what Pyruvate kinase

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splice isoform PKM2

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competitive inhibitor of PKM2

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phospho-tyrosine

produced by growth factor signaling via tyrosine kinases

when bound, releases activator fructose-1,6-bisphosphate

buildup of upstream stuff → glucose diverted into lipid synthesis

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how's PKM2 involved in response to oxidative stress

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Cysteine is oxidised by H2O2

decreases PKM2 activity

decreased glycolysis flux

diverted into PPP

increased phosphoenolpyruvate inhibits trioseposphate isomerase

increases PPP more

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PGAM1 does what

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Phosphoglycerate mutase 1

3-phosphoglycerate to 2-phosphoglycerate

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Ox PPP starting G6P to nucleotides

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Glucose 6 phosphate → 6-phosphogluconate →(6Phosphogluconatemutase) →Ribose 5 phosphate →nucleotides

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PPP starting 3-phosphoglycerate

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3-Phosphoglycerate →(3-phosphoglycerate dehydrogenase)→p-PYR→serine→glycine→nucleotides

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How's PGAM1 expression regulated

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negatively by p53

Front 89

PGAM1 regulating biosyntheis

Back 89

•regulates 3-PG and 2-PG levels

•3-PG inhibits 6-Phosphogluconatedehydrogenase (no nucleotide synthesis )

•2-PG activates 3-Phosphoglycerate dehydrogenase (activates nu biosynthesis???) - provides feedback control to increase pathway through serine

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how they studied the intracellular environment

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cryogenic electron tomography

vitrified sample

thick (0.25-1.5 µm)

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thicknes of electron microscopy samples

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50-100nm

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resolution limit of cryo-TM

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4-5 nm

protein complexes >400kDa

2nm prospect

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how's free cytosolic [ADP] mesured

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by measuring creatine kinase substrates:

Creatine-P2- + MgADP- + ATP ↔ Creatine + MgATP2-

And then Rearrange Keq stuff

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why's cytosolic ADP low in muscles

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compartmentation by binding: ADP binds actin

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ways of compartmentation

Back 95

by binding

in cellular organelles

metabolite gradients

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what's used as a probe for intracellular ATP concentration

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Firefly luciferise

Km=mM for ATP luminescence

used to report local glu concentration

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what promotes protein-protein interaction

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high P concentrations

solvent exclusion effects

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What's substrate channelling

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enzymes complex, substrates pass between enzymes w/o equilibrating

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advantages of substrate channelling 6

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high fluxes with low intermediates

•Intermediates isolated from side reactions

• unstable intermediates protected

• unfavourable equilibria avoided

•Faster response - reduction of lag times in transients between steady states

• regulation of flux

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evidence for substrate channelling

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studies on isolated and non-isolated enzyme systems

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heksokinases in brain and muscle

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HKI brain

HKII muscle

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coordination of initial step of glucose metabolism w/ mit ox ph

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glucose is phosphorylated using mit-generated ATP by hexokinase:

Incubate w/ radact P and ATP: G6P will be rad active (with isolated mitochondria)

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positioning of Hexokinase

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bound to mitochondria,

if displaced (by cell-permeable peptide containing HKII binding motif) causes mitochondrial membrane depolarisation, mitochondrial swelling and structural disruption of the cardiac tissue. Control peptide without effect

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where's creatine phosphate shuttle?

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creatine kinase is associated with myofibrillar M-band, coupled to the myofibrillar Mg2+ ATPase

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3 points on why the localisation of the enzyme is important for the creatine kinase system

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• acts as energy buffer maintaining concentrations of ATP, ADP and H during muscle contractions

•maintain high local ATP/ADP at myofibrils - rapid ATP utilisation

•energy shuttle - PC is better for diffusion than ATP/ADP

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Why's Phosphocreatine better for energy transport than ATP

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present at higher (local?) concentration

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How's PC 'shuttle' hypothesis proved

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mouse creatine kinase gene disrupted

Muscles adapted by producing more bigger mitochondria - reduced diffusion distance

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Drosophila flight muscle localisation of stuff

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•glycerol 3-phosphate dehydrogenase (GPDH-1)

•localised to the Z discs and M lines in D flight muscle

•colocalisation of aldolase and glyceraldehyde 3-phosphate dehydrogenase

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Drosophila Gpdh null alleles

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No localisation of GAPDH and aldolase

•localistion restored by plasmid expressing GPDH-1 (it has 3peptide at C term for localisation

•localisation NOT restored by expression of GPDH-3 (w/o the thripeptide)

•GPDH-3 had normal levels of the enzymes, but couldn't fly

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localised glycolysis in vascular endothelial cells

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•controls vessel sprouting

•PFKFB3 has hiher kinase activity than P2ase

•favours production of F2,6P2 - activation of glycolysis

•relocated from perinuclear cytosol to lamellipodia protrusions in motile endothelial cells

•co-locates with F-actin and other glycolytic enzymes

•Mitochondria couldn't fit into the protrusions

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substrate channelling in vivo - examples

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•Hexokinase

•Creatine phosphate 'shuttle'

•GPDH-1 localisation in Drosophila

•vessel sprouting in vascular endothelial cells

•Aldolase mobilation from actin

•Glycogen synthase and glucokinase on the move

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glycogen synthase possitioning in liver

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generally scattered

migrates to the cell periphery and co-localises with actin when glucose's present

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glucokinase positioning in lvier

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with high glucose, glucokinase translocated from nucleus to periphery with glycogen synthase

glycogen first deposited in the cell periphery

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glucokinase vs hexokinase vs glu-6-p in liver

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gluK & hexK raise G6P conc

but only gluK increases glycogen synthesis, activates glycogen synthase more (deP more)

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Archean oceanic environment is good for 3 things

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•stabilising sugar phosphates

•↑specificity of non-enzymatic pyruvate formation

•accelerated glycolysis- and pentose phosphate-like non-enz reactions

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metabolic network?

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scale-free network where few hubs link it together

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degree of interconnectivity characterised by

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network diameter

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define network diameter

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shortest biochemical pathway averaged over all pairs of substrates

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approximate network diameter

Back 119

3

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advantage of scale-free metabolic network

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allow perturbations to be rapidly transmitted

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elementary flux modes

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smallest sub-network enabling the metabolic system to operate at steady state

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network robustness vs number of elementary modes connecting metabolites

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increases - if something malfunctions, another enzyme can take over from another elementary mode

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yeast: main network robustness mechanism

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network redundancy through replication of the genes

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Isoenzymes vs robustness

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appear where high flux is needed

duplicates permit selectively advantageous increase in flux rates

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pathway is usually in what?

Back 125

dynamic steady state

Front 126

methods for identifying flux controlling enzymes

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•enzymes with low Vmax

•enzymes catalysing far from eq reactions (mass action ratio/Q<<Keq)

•Look for cross-over points: [reag]↑ and [prod]↓

•Control points at the start of pathway or after branch points

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metabolic control analysis

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relates flux and intermediary metabolite levels to the activities of the component enzymes

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Flux control coefficient of an enzyme

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fractional change in pathway flux/fractional change in enzyme concentration

sum of all in linear pathway =1

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flux control coefficients' measurements in mit ox ph

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titrating enzymes' activities with inhibitors

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inhibitor of adenine nucleotide translocase

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carboxyatractyloside

Front 131

experiment to measure adenine nucleotide translocase flux control coefficient: two points to note

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flux control is distributed with all components showing some control

•the distribution of control changes depending on the rate of respirtaion