meds2003 metabolism

catabolic pathway

processes that break down larger molecules into smaller ones to release energy. generally extract H/e- which are delivered to ETC to harness energy, or substrate level phosphorylation can produce ATP directly

anabolic pathways

processes that synthesize larger molecules from smaller ones, requiring an input of energy (ATP) or reducing ower (NADPH, NADH)

AMP

contributes the most to cells energy state, small AMP change = large change overall. high AMP = low ATP levels

5mM ATP

amount of ATP needed by cells to survive, if below 3mM cells die

kinase

catalyses a phosphorylation reaction (addition of a phosphate)

phosphatases

catalyse dephosphorylation reactions (remove phosphate)

phosphorylases

catalyse a phosphorolysis reaction (using a phosphate to break/lyse other substrates)

synthase

catalyse condensations, no ATP needed

synthetases

catalyse condensations, with ATP (or GTP, CTP, TTP)

enzymes role

reduce activation energy for a reaction, don’t change equilibrium

dehydrogenase

catalyses a redox reaction, usually involves NAD+/FAD as cofactors, named for substrate which is oxidised.

ATP Hydrolysis

ATP → ADP or AMP, 1 or 2 phosphates removed, releases energy for cellular processes. often coupled to provide energy to unfavourable reactions.

relative rates of catabolic and anabolic pathways

regulated by energy state of the cell. energy charge = ATP + ½ ADP / ATP + ADP + AMP. AMP biggest regulator of energy state. energy charge = ) = more catabolic to make ATP, energy charge close to 1 = more anabolic to use up excess ATP.

NAD+/NADH

NAD+ = oxidised form, NADH = reduced form

NADPH

variation of NADH carrier, contains a phosphate. produced in pentose phosphate pathway, often used for lipogenesis.

Coenzyme A

great for acyl groups, can trap things within cells (eg, fatty acids) by bonding to it. acetyl CoA → reactive group is esterified. the pantothenate unit of the cofactor can recognise many enzymes.

fuel oxidation - general

1. H+/e- are removed from fuels and the fuels are broken into 2 carbon chunks of acetate, which is carried around by acetyl CoA (at this point carriers are full - FADH2, NADH).

2. H+/e- are removed from acetyl Coa in Krebs cycle, forming 2CO2.

3. electron transport chain - hydrogen + oxygen react and liberates energy which is captured, proton gradient forms, protons flow through ATP synthase, subunits spin and convert ADP → ATP.

fuels

1. fatty acids - beta oxidation

2. glucose/carbs - glycolysis

3. amino acids/proteins - several pathways

beta oxidation - overview

1. fatty acids trapped in cytoplasm as fatty acyl-CoA

2. transported to mitochondria by carnitine

3. stripped of electrons + protons by FAD/NAD

4. lose acetate chunks

5. cycle repeats

glycolysis - overview

1. glucose is phosphorylated

2. split into two 3C chunks (krebs)

3. both chunks oxidised

4. NAD reduced to NADH

net gain = 2 ATP, 2 pyruvate, 2 NADH

krebs cycle - overview

1. acetyl-CoA converted to citrate

2. stripped of electrons + protons

3. oxidised to CO2

4. converted to oxaloacetate

5. cycle repeats

inner mitochondria membrane

impermeable to protons, hence ATP synthase

proton pump “broken”

when the proton gradient is too high?

type 1 muscle fibres

aka, red/slow muscle fibres

good blood supply

many mitochondria

type 2 muscle fibres

aka white/fast

many contractile filaments

low blood supply w/ few mitochondria

PDH

pyruvate dehydrogenase, removes 3rd carbon of pyruvate for beta oxidation, allowed ac-CoA (2C chunk) to enter krebs

inhibition of PDH

acetyl-CoA inhibits……

fuel for initial stages of exercise

glucose, after a few mins - switch to fatty acids

moderate exercise fuel

start w/ fatty acids, after few minutes, enzymes reach Vmax, PDH inhibition by acetyl CoA removed, glucose used again

strenuous exercise fuel

eg, sprinting, glucose + fatty acid used initially, one glycolysis reaches max capacity, muscle glycogen stores used.

glycogen use during exercise

problems = lactate buildup and very low ATP generation

solution = creatine phosphate → creatine (provides 15 mM ATP)

effects of lack of O2

1. ATP synthesis stops

2. rate of proton pumping stops

3. proton gradient dissipates

4. rate of O2 consumption stops

fatty acid forms

glycerol, free fatty acids, triglycerides

storage of fatty acids

stored mostly in fully reduced form as triglycerides, they are hydrophobic and energy dense stores, cannot be used as fuel by brain

glucose storage

glucose stored as glycogen, reasonably reduced form, hydrophilic, low stores and may be used by all tissues. glycolysis = no O2 required, wholly cytosolic process, fast but inefficient

use of amino acids as fuel

only when ABSOLUTELY necessary, many pathways unique to the aa. channel into pyruvate, acetyl-CoA, or krebs. inefficient. stores = 5-10 kg

muscle action

muscle contractions use ATP, filaments slide across each other at rest and still use ATP at rest. sprinting muscle = 5 mM ATP / second

onset of exercise

immediate increase in use of ATP as substrates available increase (ADP), ETC rate increases to restore ATP = coupling process

coupling - in presence of exercise

rate of ATP synthesis is equal to rate of ATP use. ATP turnover is much higher than ATP stores even at rest

ATP stores

1 g / kg body weight

ATP turnover

1 kg / kg body weight

glucose transport

1. muscle contractions (during exercise) cause GLUT transporters to move from golgi to cell surface to allow gluocose movement.

2. glucose is trapped in cell by adding a phosphate via hexokinase

3. lowers blood glucose

4. glycogen released to restore it (in response to low insulin, high glucagon) to 5 mM

AKA = glucose homeostasis

trapping fatty acids in cytoplasm

1. in blood FA bound to albumin

2. FA enters cell via passive diffusion, kept inside by fatty acid binding protein (FABP)

3. trapped by CoA - fatty acyl CoA synthetase attaches the CoA to fatty acids (costs 2ATP)

albumin

most abundant blood protein

transport of FA

in cytoplasm - carnitine acyl transferase (CAT1) replaces CoA with carnitine to enter mitochondria

in mitochondria - CAT2 replaces carnitine with CoA

reduction of FAD → FADH2

form a C=C bond, involves dehydrogenase

reduction of NAD → NADH

forms a C=O bond/group, involves dehydrogenase

products of beta oxidation

each round = 1 acetyl CoA, 1 NADH, 1 FADH2

eg - 16C chunk = 7 rounds = 8 acetyl CoA, 7 NADH, 7 FADH2

GLUT1 Location

in all cells

GLUT4 location

muscle and adipose tissue, insulin-dependent

GLUT2 location

liver and pancreas, does not depend insulin

hexokinase

converts glucose to G6P to trap in cytoplasm

inhibited by G6P, low Km

glucokinase

converts glucose to G6P to trap in cytoplasm

not inhibited by G6P

high Km for glucose (not easily saturated)

citrate

formed by combining acetyl-CoA with oxaloacetate

how does NADH enter mitochondria

1. glycerol 3 phosphate shuttle

2. malate aspartate shuttle

glycerol 3 phosphate shuttle

1. NADH passes e- and H+ to dihydroxyacetone phosphate which generates glycerol 3 phosphate

2. delivers to mitochondrial G3P dehydrogenase

3. transfers e- and H+ to FAD which becomes reduced to FADH2

malate aspartate shuttle

1. NADH passes e- and H+ to oxaloacetate to form malate

2. malate enters mitochondria and regenerates oxaloacetate

3. also passes e- and H+ to NAD which becomes reduced to NADH

which complexes pump H+ in ETC

1 - pumps 4 H+

3 - pumps 4 H+

4 - pumps 2 H+

what is ubiquinone?

intermediate electron carrier in the ETC (between 1/3 or 2/3 proteins)

accepts e-, then passes them off,

it is both reduced and oxidized

proton releasing reactions

cytoplasmic side of mitochondria

proton consuming reactions

matrix side of mitochondria

how many protons need to pumped to generate 1 ATP

3 protons

ATPase mechanism

1. 3 protons enter

2. gamma subunit rotates

3. beta subunit of F1 changes conformation - accepts ADP and phosphate, reacts them to produce ATP, released ATP

lactate fuelled gluconeogenesis products

30 g / day glucose

gluconeogenesis de novo

90 g / day glucose, from glycerol (lipolysis), carbon backbone of aa (from proteolysis)

glycogen structure

1. glycogenin protein

2. branching chains of glucose (12-14 glucose long)

3. non reducing ends (where new glucose are added)

4. alpha1,4 and alpha 1,6 glycosidic bonds

early starvation (first 24 hrs)

• increase in glucagon

• glycogenolysis - breakdown of glycogen

• lipolysis - breakdown of fat

• used to produce glucose for brain

glycogenolysis during early starvation

1. phosphorylase removes a glucose from glycogen to form G1P

2. G1P is converted to G6P

3. G6Pase dephosphorylates G6P to form glucose

4. glucose exits into blood via GLUT2

events when blood conc. increases

1. glucagon binds to receptors on liver cell

2. activates cAMP

3. activates protein kinase A (PKA)

4. activates (phosphorylates) phosphorylase kinase

5. activates (phosphorylates) glycogen phosphorylase

result - glycogen → G1P

aka phosphorylation cascade

effect of high glucagon on WAT - early starvation

glucagon binds to receptors

• activates cAMP

• activates PKA

PKA activates (phosphorylates)

• perilipin (shell around fat), allows HSL to interact w/ fat

• hormone sensitive lipase which hydrolyses triglycerides

result = FA utilized to create acetyl CoA, fuels krebs + inhibits PDH to conserve glucose

activity of PDH

active → dephosphorylated (by PDH phosphatase) by insulin

inactive → phosphorylated (by PDH kinase) by acetyl-CoA

late starvation processes

1. liplysis

2. proteolysis

3. gluconeogenesis

when beta oxidation rate is at max

• formation of ketone bodies by fusing 2 molecules of acetyl CoA

• fate of ketone bodies - used in tissues (provides up to 90g/day glucose), used in krebs (splits to form acetyl CoA)

ketone body disposal

1. excreted in urine

2. spontaneously decarboxylate to acetone (useless) and bicarb

proteolysis in late starvation

problems

1. need to transport aa to liver without being used by other tissues

2. need to convert amine groups into urea (non-toxic) as ammonia is toxic

urea cycle

1. passes amine groups to acceptors to form alanine/glutamate/aspartate

2. which then enter liver to produce urea

3. skeleton left will be used as substrate for gluconeogenesis

costs of using aa carbon skeletons

1. costs ATP to synthesize proteins

2. costs ATP to dispose amine groups

3. only glucogenic aa can be used to produce glucose

substrates for gluconeogenesis

glycerol, aa, lactic acids

glucogenic vs. ketogenic aa’s

glucogenic

• can be converted into glucose via gluconeogenesis

• all except leucine + lysine

ketogenic

• can only be converted into acetyl-CoA

coupling

rate of fuel oxidation is matched to rate at which ATP is used to keep constant levels of about 5mM in cell, demand driven system. rate of fuel O2 relates to energy expenditure

uncoupling

system breakdown → when H+ can’t enter via another method (not ATP synthase), proton gradient disappears and no ATP made

• can be caused by DNP

DNP - dinitrophenol

• hydrophobic when protonated

• moves freely across membrane

• weak acid

• H+ loss doesn’t change polarity as -ve delocalised (resonance stabilized)

• dissapates proton gradient

• massive weight loss + heat production

• used as weight loss agent but toxic

natural uncoupler (UCP-1)

• uncoupling protein 1

• thermogenin - generates heat

• found only in brown adipose

• high levels in neonates

used for = heat gen

hormones controlling UCP-1

noraderenalin binds to P3-receptors, stimulating fatty acid release, this opens protein channels to all H+/e- to enter

electron transport chain overview

• 4 complexes embedded in inner mitochondrial membrane (I, II, III, IV)

• complexes made up of proteins and various functional groups - all have a prosthetic group (transports H+/e-), proteins arranged so - H+ expelling on outside, H+ consuming on matrix side

• about 10 H+ pumped for each NADH

ETC NAD

• donates H+/e- to complex I

• NAD+ accepts a hydrogen ion and 2 e'- so 1 H+ left in solution

• NADH higher absorbance than NADH

ETC FAD

• inside complex II, accepts and donates H+

UQ (ubiquinone)

• very hydrophobic

• picks up Hs from complex 2

• passes Hs to complex 3

cytochrome C + iron

• Cyt C takes e- from 3 to 4

• Cyt C has prosthetic group containing iron

• changes from ferrous to ferric as loses and accepts e-

• does not carry H+

• Fe3+ → Fe2+

Proton Pumping

• when different carriers exchange H+ and e-, H+ can be taken up of released

• orientation of uptake/release allows net pumping of H+ into cytoplasm

results

• 10 H+ per NADH

• 6 H+ per FADH2

mitochondria overview

• protons are pumped across inner mitochondrial membrane as electrons flow through the respirtory chain

• high H+ conc = intermembrane space (+ve)

• low H+ conc = matrix (-ve)

outer mitochondrial membrane has large pores allowing big proteins to pass

movement of NADH

• NADH enters cytoplasm via glycerol-3-phosphate (6 H+ as skips complex 1)

• or via malate aspartate (no H+ loss)

free radicals

ETC is a free radical producer

• e- in UQ pool react w/ molecular O2

• free radicals damage DNA

• less likely to form if complex 3 is vacant

ATP synthase subunits

• as protons enter the y subunit rotates which causes conformational changes in the 3 B and 3 A subunits

• beta subunit - accepts ADP + Pi, reacts together and then releases

• every time 3 protons come in, the entire complex spins once = 1 ATP gen

other uses for proton gradient

• not every H+ in FADH2 etc contributes to ATP gen

• exchange of 1 H+ allows 1 ATP to exit matrix for use elseware

• also need to allow phosphate entrance

• inhibitors and acceptors can alter ETC

adipose lipolysis

• glucagon ^ cAMP

• ^ cAMP ^ PKA activtiy

• PKA phosphorylates hormone sensitive lipase (activates it) and also perilipin (surrounds fat vacuole)

• allows HSL to interact w/ fat, releasing it into blood stream

• conserves glucose during starvation

cori cycle

glucose recycling

muscle glucose → pyruvate → lactate → glucose (via glucaneogenesis) → glucose in bloodstream

glucaneogenesis

converts lactate to glucose

proteolysis

• after few hours of glucose < 5mM, insulin secretion stops (starvation)

• lipolysis stimulation stops

• widespread proteolysis (proteins → aa)

• mainly occurs in muscles

• skeletons of aa (aa without amine) used in neogenesis

• amine group must be toxified

• aa’s transported to liver

processing aa’s

• channel amine groups to 3 aa’s (alanine, glutamate, asparate)

transport via:

• pyruvate - alanine

• alpha-keto glutarate - glutamate

• oxaloacetation - asparate

all enter urea cycle to be detoxified

resulting alpha keto acids used in gluceoneogenesis