Metabolism, Nutrition & Endocrinology (Part I)

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Georgetown SMP '14 Molecular & Cellular Physiology

Purines

Adenine & Guanine

Pyramidines

Cytosine, Thymine & Uracil

Guanine

replaces adenine in ATP to make GTP

Thymine

in DNA

Uracil

in RNA

ATP

Major energy source in cell, but not sole source

*hydrolysis of phosphate bond releases heat → energy*

structure:
∙three phosphate groups with phosphoester bond b/w alpha phosphate and adenosine group
∙beta and gamma phosphates attached by phosphoanhydride bonds
→require lots of energy
→when broken, lots of energy released

Harnessing chemical energy

When bonds are broken, energy is released, either in form of heat or in terms of work done by system

Nucleophilic Attack of Phosphate

Nucleophilic attacks help drive reactions in one direction in cell, since enzymes aren't always picky about which direction rxn goes

Nucleophile attacks, leads to intermediate state, phosphate group then transferred and leaving group leaves → energy transferred to nucleophile
**Key: ΔG=ΔH-TΔS, rxn needs to be energetically favorable
∙just because phosphate linkage may not have enough energy to transfer phosphate doesn't mean rxn can't happen → if rxn can absorb enough heat it can happen, entropy helps too

ATP and nucleophilic attack

ATP phosphate can be attacked at any bond by nucleophiles

Possible products: Nucleophile bond to gamma phosphate, bound to gamma and beta phosphates, or bound to alpha phosphate and adenosine

NAD⁺ and NADH

electron carriers of cell

Oxidized: NAD⁺
Reduced: NADH

composed of AMP (lower ring with adenine) plus a nicotinamid base (upper ribose with red ring)

NADP⁺ is same molecule with extra phosphate group added to AMP

both can be reduced, but NAD⁺ reduced in catabolism, NADPH oxidized during anabolism
→done to keep high energy electrons separate

rxn that reduces NAD⁺ to NADH is limited by acidic conditions

Redox Reactions

Most energy is cell comes from high energy electrons being shuffled around so ATP can be more or work can be done, aided by oxidation and reduction
→burning of fuel takes lots of fuel, cells break down into smaller chunks via oxidation
→building of fuels (easier) done w/ reduction rxns

*often a H atom may be referred to as electron b/c it is typically donating its electron to another atom to fill shell (i.e. oxygen) → NAD⁺ to NADH

Catabolism

breakdown of more complex substances into simpler ones with release of energy

Anabolism

synthesis of more complex substances from simpler ones

Catabolism vs. Anabolism

Energy derived from flow of electrons to oxygen

Addition of hydrogen is actually a reduction

new molecule requires a lot of energy to remove hydrogen once added

Main fuel sources

Glucose (difficult to store) & saturated fatty acids (easy to store, preferred)

both can be catabolized for OxPhos, only glucose can be catabolized anaerobically (glycolysis)

both need to become acetyl-CoA to go through citric acid cycle, both will send electrons via NADH and FADH₂ to electron transport chain while acetyl group is completely oxidized

Mitochondria

Most likely came from bacterial infection of eukaryote and eventually had symbiotic relationship

have own DNA/ribosomes, act like bacteria

outer membrane is permeable to everything, inner membrane is impermeable to everything
→important b/c electron transport chain proteins are on inner membrane and shuttle H⁺ ions out of matrix and into intermembrane space

matrix is where citric acid cycle takes place

Electron Transport Chain

Complex I → coenzyme Q (ubiquinone) → Complex III → soluble carrier protein cytochrome c → Complex IV → oxygen
10 protons total**

Complex II (succinate dehydrogenase) → coenzyme Q → complex III → cytochrome C → complex IV → oxygen
*6 protons total*

Alpha Amino Acids & Alpha Keto Acids

core backbone structure is identical, simple changes between side groups (via amination/deamination) switches b/w 2 forms

Routes of entry to ETC

depends on which electron carrier is used:
∙NADH → electrons to Complex I
∙Succinate → electrons to Complex II

Acceptor Of Reducing Equivalents from NADH in ETC

NADH hands off two electrons to FMN in complex I, makes NAD⁺ and H⁺

w/in complex I, electrons FMNH₂ transfer to iron-sulfur center

FMN

Flavin mononucleotide

cofactor that is covalently attached to one of amino acids making up complex I

triple ring structure

accepts 2 electrons to become FMNH₂
→electrons bound to FMN will have lower energy than thouse bound to NADH

when bound to adenylate nucleotide instead of amino acid, considered flavin adenine dinucleotide (FAD)
→both derived from riboflavin (vitamin B₂) while NAD is derived from niacin (vitamin B₃)

Iron-Sulfur Center

Iron is chelated by elemental or cysteine-bound sulfurs

Iron can accept electrons in Fe³⁺ state to become reduced Fe²⁺, can easily donate electrons to return to oxidized state
→electrons from FMNH₂ transfer to Fe-S center

abundance of Fe-S centers in inner membrane of mitochondria is actually what gives dark appearance to meat b/c tissue is slow twitch muscle, which uses fatty acids as major metabolic fuel
→fatty acids broken down by OxPhos in mitochondria

Coenzyme Q

lipid soluble cofactor that contains benzoquinone ring and hydrophobic isoprenoid chain
→side chain responsible for solubility w/in lipid membrane
→benzoquinone reduced upon accepting electrons from coenzyme Q

accepts electrons from Complex I's iron-sulfur center in ETC

Coenzyme Q transfers electrons to complex III in ETC

Complex III pumps out 4 more protons into inner mitochondrial space

Cytochrome C

Free protein in intermembrane space
→all contain heme - hold iron in place
**unlike hemoglobin, iron's role here is to hold electrons, i.e. in complex I

transfers electrons from complex III to complex IV in ETC

Energy from protons pumped by ETC is harnessed for ATP production

As protons are pumped across membrane, local gradient forms
→both a chemical potential (b/c cation accumulation results in low pH) and electrical potential (because of charge)
→potentials are local (not huge pH change occuring throughout entire matrix)

energy expended to create potentials → when proton returns to mitochondrial matrix, energy is released
→energy harnessed by F₁ ATPase to form phosphoanhydride linkage between ADP and inorganic phosphate

F-Type ATPase/ATP Synthase

F₀ domain - integral membrane protein
F₁ domain -catalytically active portion, peripheral membrane protein

Typically seen working in reverse direction → ATP hydrolyzed to provide energy to pump protons
→in inner mitochondrial membrane, however, proton gradient drives ATPase in opposite direction so ATP is produced
→loss of gradient = ATPase reversal, i.e. asphyxiation

Transport of ATP, ADP, Pi in ETC

Adenine nucleotide translocase (antiporter) exchanges ATP for ADP across inner membrane (brings in ADP for ATPase to use)

Phosphate translocase (symporter) uses proton gradient to bring phosphate into matrix from intermembrane space to provide phosphate for ATPase

Once ATP is in intermembrane space, it will freely cross outer membrane to cytosol

Stiochiometry of OxPhos

4 protons to make an ATP

# of protons pumped per pair of electrons:
∙starting from NADH: 10
∙starting from succinate: 6

Net yield: 2.5 ATP per NADH oxidized, 1.5 per succinate

Regulation of OxPhos

provides most of ATP produced in aerobic cells

complete oxidation of glucose (glycolysis, decarboxylation of pyruvate to acetyl CoA, TCA, electron delivery to ETC) yields 30-32 ATP molecules

Rate of respiration and OxPhos limited by level of ADP
→when not limited:brown fat

Hydrolysis of ATP by ATP synthase limited during ischemia by IF₁ inhibitor

ETC and Ischemia

In ischemia, ETC slows because there is no oxygen to accept electrons after complex IV
→would expect gradient to collapse w/o electron flow through ETC but IF₁ inhibitor prevents ATPase from functioning in reverse and hydrolyzing ATP

with loss of OxPhos, anaerobic glycolysis kicks in
→end products: pyruvate and lactic acid (negatively charged), resulting in decreased pH
→metal cations such as K⁺ or Na⁺ will exit mitosol in exchange for H⁺ along inner membrane (once in intermembrane space, freely cross outer membrane to cytosol, positive charge helps neutralize acidic pH)
→as mitosol loses cations, becomes acidic, activating IF₁, which binds to F₁ on ATPase to shut down function

Brown Fat

type of adipose tissue that is rich in mitochondria → dark appearance from iron

uncoupling protein (simple protein channel) present in cell's mitochondria
→allows protons to return back to mitosol but energy si released as heat instead of being captured in ATP
→proton gradient now uncoupled from ATP respiration
→some poisons take advantage of this mechanism by using proton ionophores, surround proton to allow it to cross inner membrane w/o passing through F-Type ATPase, which collapses gradient, energy from gradient released solely as heat
*does not block respiration, only productive use of gradient for ATP synthesis*

Cyanide Poisoning

Cyanide inhibits cytochrome oxidase (Complex IV) step of ETC

binds Fe³⁺ on one of hemes
→once bound, iron can no longer accept electrons, occluding electron flow

inhalation of amyl nitrate or IV infusion of NaNO₂ are treatments
→converts oxyhemoglobin to methemoglobin
→oxygen delivery not compromised b/c this affects only small portion of total hemoglobin
→small portion of methemoglobin provides an Fe³⁺ to cyanide which it will preferably bind to over iron in cytochrome A

Glycolysis

process that metabolizes glucose so we can derive energy from it

anaerobic rxn

occurs in cytoplasm
→pyruvate decarboxylation, TCA, ETC all w/in mitochondria

also functions to produce intermediates that are important starting points in anabolic rxns w/in cell
→major mechanism en route to FA production (need pyruvate →acetyl-CoA→citrate)
→many enzymes also involved in gluconeogenesis (generation of glucose through non-carbohydrate sources)

2 phases: preparatory phase & payoff phase

Glucose is major fuel in skeletal muscle

especially in fast twitch

anaerobically converts glucose to pyruvate in cytosol, which is converted to lactic acid

after release from cytosol into blood stream, lactic acid returns to liver to be converted back to glucose

in slow twitch, w/ aerobic conditions, pyruvate instead sent into mitochondria matrix to be converted to acetyl CoA, continue citric acid cycle

Preparatory phase of glycolysis

Glucose is converted to two phosphorylated carbon molecules → glyceraldehyde-3-phosphate and dihydroxyacetone phosphate

ATP beta-gamma phosphate bond hydrolysis is coupled to glycolysis in order to form low energy phosphate compounds (G6P, G3P) → made into high energy phosphate compounds through pathway

in this phase, must be investment of ATP before eventual generation of ATP

Payoff phase of glycolysis

hydrolysis of high-energy phosphate bonds yields new ATP

get 2 ATP we invested back, plus 2 additional ATP

relatively small payoff

also get NADH

Key questions about glycolytic pathway and its role in fuel metabolism

In thermodynamic terms, what drives the forward rxn of glycolysis?

How are enzymes in pathway regulated to control the rate of glycolysis, and what purpose does this serve?

What is the chemical strategy used to harness energy from glucose, and what advantages and limitations does this pose physiologically?

Steps of Glycolysis

1. Hexokinase
2. Phosphohexose Isomerase
3. Phosphofructokinase-1 (PFK1)
4. Aldolase
5. Triose Phosphate Isomerase
6. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
7. Phosphoglycerate Kinase
8. Phosphoglycerate Mutase
9. Enolase
10. Pyruvate Kinase

Glycolysis Net Yield

Glucose + 2 ADP + 2 Pi + 2 NAD⁺ → 2 pyruvate + 2 ATP + 2H⁺ + 2 H₂O

1st Rxn of Glycolysis: Hexokinase

[Glucose + ATP → glucose-6-phosphate + ADP]

first priming rxn

*hexokinase phosphorylates 6th position of glucose, forming G6P, ATP is hydrolyzed*

provides large negative ΔG that drives forward rxn (ΔG = -16.7 kJ/mol)

irreversible

phosphorylation retains glucose in cell
→only glucose can pass through membrane transporters (GLTS), G6P will be retained in cell for fuel in glycolysis or made into glycogen as storage

2nd rxn of glycolysis: phosphohexose isomerase

[G6P ←→ Fructose-6-Phosphate (F6P)]

not very important step

has positive ΔG, therefore reversible, rxn direction is driven by concentration ratios
→subsequent rxn rapidly removes product, favoring forward rxn

Free Energy Level of Phosphorylated Compounds in Glycolysis

3rd step of glycolysis: PFK1

[F6P + ATP → Fructose-1,6-Bisphosphate (F1,6bP) + ADP]

F6P further phosphorylated by PFK1 to F1,6bP coupled to hydrolysis of beta-gamma phosphate bond of ATP

highly negative ΔG, irreversible

*FIRST COMMITTED STEP*
→once F6P gets phosphoryated to F1,6bP it has no choice but to go down glycolytic pathway and be metabolized

B/c this step is checkpoint for glycolytic pathway, activity of PFK1 is heavily regulated

4th step of glycolysis: Aldolase

[F1,6bP ←→ dihydroxyacetone phosphate (DHAP) + glyceraldhehyde-3-phosphate (GAP)]

F1,6bP cleaved into two triose sugars
→only GAP moves forward in glycolysis
→DHAP can be converted to GAP in next rxn, or used as intermediate in FA synthesis

Rxn has very positive ΔG, readily reversible
→main driving force is low product concentration b/c product is rapidly depleted by subsequent rxns

5th step of glycolysis: Triose Phosphate Isomerase

[DHAP ←→ GAP]

DHAP isomerized to GAP to continue in glycolysis

last step in preparatory phase of glycolysis

6th step of glycolysis: glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

[GAP + Pi + NAD⁺ ←→ 1,3-Bisphosphoglycerate (1,3bPG) + NADH + H⁺]

1st step in payoff phase b/c formation of high energy phosphate compound (1,3bPG)
→acyl phosphate bond very unstable, has high phosphoryl group transfer potential (can be readily transferred to ADP)

Pi comes from inorganic phosphate, NOT ATP

NAD⁺ reduced as it accepts electron GAP
→must be regenerated for rxn to occur again

reversible

accumulation of lactic acid and H⁺, decrease in pH from physical exertion makes pathway less effective, favors reverse rxn

7th step of glycolysis: Phosphoglycerate Kinase

[1,3bPG + ADP ←→ 3-phosphoglycerate (3-PG) + ATP]

high energy acyl phosphate bond in 1,3-bPG broken down to form 3-PG
→phosphoryl group transferred to ADP to form ATP

PAYOFF PHASE WHERE ATP FORMED

reversible, despite highly negative ΔG
→reversed in gluconeogenesis

enzyme called kinase for adding phosphate group in reverse rxn

Oxidoreductases

enzymes that catalyze transfers of electrons (hydride ions or H atoms)

Transferases

enzymes that catalyze group-transfer reactions (.ie. methyl group or phosphoryl group)

Hydrolases

enzymes that catalyze transfer of functional groups to water

Lyases

enzymes that catalyze addition of groups to double bonds, or formation of double bonds by group removal

Isomerases

enzymes that catalyze transfer of groups within molecules to yield isomeric forms (from one part of a molecule to another part of same molecule, basically just rearranging)

Ligases

enzymes that catalyze formation of C-C, C-S, C-O and C-N bonds by condensation reactions coupled to ATP cleavage (energy dependent reaction, usually uses ATP, but sometimes uses other high energy cofactors)

Kinases

phosphorylate proteins with ATP as phosphate donor

Desphosphorylases

enzymes responsible for removing phosphoryl group

8th step of glycolysis: phosphoglycerate mutase

[3-PG ←→ 2-phosphoglycerate (2-PG)]

unimportant

phosphate group moved from 3rd carbon to 2nd

reversible, positive ΔG

9th step of glycolysis: enolase

[2-PG ←→ phosphoenolpyruvate (PEP) + H₂O]

forms another high energy phosphate compound, PEP, w/ elimination rxn forming double bond, releasing water

10th step of glycolysis: pyruvate kinase

[PEP + ADP → pyruvate + ATP]

last step

very important b/c high energy phosphate in PEP is transferred to ADP

irreversible, highly negative ΔG
→bypassed in gluconeogenesis

Regeneration of NAD⁺

Aerobic regeneration: occurs in mitochondria, produces high yield ATP, but NADH cannot pass through inner membrane of mitochondria and requires electron carrier for transport, making process slow

Anaerobic regeneration: occurs in cytosol, where pyruvate converted to lactate by lactate dehydrogenase (LDH), NAD⁺ rapidly generated, can be fed back into GAPDH step of glycolysis
→problem is that lactate accumulation can create oxygen debt, acidosis
→lactate reconverted to pyruvate in liver

Regulation of PFK1

if surplus of energy or products in cytosol, want to slow down glycolysis

if we have shortage of energy indicated by high levels, of AMP, ADP, or high concentrations of upstream metabolites (f2,6bP) we want to speed up glycolysis

F2,6bP

allosteric stimulator of PFK1

phosphofructokinase-2 (PFK2) constitutively on in muscles, converts small amount of F1,6bP into F2,6bP

F2,6bP is indicator of F1,6bP, indicating PFK1 needs to increase rate

Pentose Phosphate Pathway (PPP)

we are not responsible for names of enzymes/intermediates, but know significant in metabolism and major end products

Oxidative phase & non-oxidative phase

Purpose of pathway:
∙reduction of NADP⁺
∙synthesis of ribose-5-phosphate

Ribose-5-phosphate

synthesized in PPP, used for making nucleotides in DNA/RNA synthesis and cofactors (coenzyme A)

Oxidative Phase of PPP

main event is reduction of NADP⁺ to NADPH while carbon from G6P is oxidized to CO₂

NADPH has important role in anabolic rxns (cholesterol, FA and lipid synthesis), also protects cells by scavenging free radicals (preventing oxidative damage)
→particularly important in RBCs (susceptible to oxygen free radical damage)
→eliminates radicals through glutathione system

Important end product: Ribose-5-phosphate used for making nucleotides in DNA/RNA synthesis and cofactors such as coenzyme A

Glutathione

tripeptide w/ cysteine in middle capable of forming intermolecular disulfide bonds b/w 2 glutathione molecules when oxidized

converted to oxidized form (GSSG) after removal of oxygen free radicals

reduced form GSH has to be regenerated using NADPH

PPP serves as way to regenerate NADPH so we can regenerate reduced form of glutathione

Non-oxidative phase of PPP

involves 5-carbon sugars (ribose-5-phosphate) exchanging carbons, leading to formation/regeneration of G6P

GAP also a product of these rxns

Pyruvate dehydrogenase complex (PDC)

contains 3 enzymes (E1, E2, E3) and requires 5 cofactors (TPP, FAD, NAD⁺, Coenzyme A, lopoic acid)

pyruvate converted to acetyl-Coa by PDC in mitochondria, where it enters TCA

attachment of CoA activates acetyl group, allows it to be transferred to oxaloacetate in TCA

TPP

thiamine pyrophosphate, vitamin B₁₂ derivative

E1 in PDC

pyruvate dehydrogenase

pyruvate decarboxylated, acetyl group attaches to TPP in hydroxylethyl form, acetyl group then transferred from TPP to oxidized lipoic acid, which is attached to Lys residue on E2

→transfer reduces lipoic acid, attached group is again acetyl form

* rxn is irreversible because CO₂ release is highly exergonic, CO₂ sequestered, unavailable after release*

E2 in PDC

dihydrolipoyl transacetylase

CoA attacks acetyl group, releasing it from lipoic acid, generating one molecule of acetyl-CoA

E3 in PDC

dihydrolipoyl dehydrogenase

rxn in E2 leaves lipoyllysine in reduced state, needs to be reozidized in order for PDC to continue functioning

→electrons first transferred to FAD to form FADH₂, then to NAD⁺ to form NADH and H⁺

CoA

made up of 3'-phosphoadenosine, diphosphate, pantothenic acid and β-mercapto ethylamine

β-mercapto ethylamine contains thiol group (-SH) that forms thioester bond with acetyl group

Regulation of PDC

regulation occurs at E1 b/c step is irreversible

product inhibition occurs when acetyl-CoA and NADH compete with NAD⁺ and CoA for binding sites, which drives rxn backwards
→makes sense b/c end products in high concentrations should shut down forward rxn

phosphorylation of E1 by pyruvate dehydrogenase kinase inactivates enzyme →covalent modification

Citric Acid Cycle

Citrate Is Krebs' Starting Substrate For Making Oxaloacetate

occurs in mitochondrial matrix, electrons harnessed delivered to respiratory chain in inner membrane

Overall rxn: Acetyl-CoA + 3NAD⁺ + FAD + GDP + Pi →2CO₂ + CoA + 3NADH + 3H⁺ + FADH₂ + GTP

circular pathway, rate of acetyl-CoA oxidation determined by amount of intermediates available
→more intermediates, faster it is oxidized
→increase [acetyl-CoA] will never promote net increase in # of intermediates
→pyruvate can promote net increase

TCA intermediates feed many anabolic rxns

Oxaloacetate can be used as starting material for glucose synthesis
→ATP from oxidation of acetyl-CoA through TCA can fuel gluconeogenesis
→if oxaloacetate depleted during glucose synthesis, TCA will slow down, so will production of useable energy

α-ketoacids → oxaloacetate and α-ketoglutarate form carbon skeletons of aspartate and glutamate, respectively
→amination and reduction of either will form corresponding amino acids, will play key role in nitrogen regulation in cell

Steps of TCA

1. citrate synthase
2. aconitase
3. isocitrate dehydrogenase
4. α-ketoglutarate dehydrogenase
5. succinyl-CoA synthetase
6. succinate dehydrogenase
7. fumarase
8. malate dehydrogenase

1st step of TCA: citrate synthase

irreversible

addition of acetyl-CoA (2C) to oxaloacetate (4C) to create citrate (6C)

large negative ΔG needed to drive rxn forward b/c there is little starting material

2nd step of TCA: aconitase

reversible

rearrangement rxn to form isocitrate from citrate
→involves elimination and addition of water to change location of hydroxyl group

3rd step of TCA: isocitrate dehydrognase

irreversible, 1st oxidation step

involves decarboxylation and oxidation to form α-ketoglutarate

has large negative ΔG

either NAD⁺ or NADP⁺ can act as electron acceptor, specific isoform of enzymes determines which is used
→one of two isoforms used, never both

4th step of TCA: α-ketoglutarate dehydrogenase

irreversible

analogous to PDC
→same cofactors, same structure
→instead of CoA being attached to acetyl group, it is attached to succinyl group → forms succinyl-CoA

large negative ΔG

5th step of TCA: succinyl-CoA synthetase

reversible

thioester linkage formed with CoA carries large amount of free energy, harnessed and used to form phosphoanhydride bond in synthesis of GTP

modest ΔG because reaction coupled to GTP synthesis

6th step of TCA: succinate dehydrogenase

readily reversible
→ΔG = 0
succinate dehydrogenase is Complex II in ETC respiratory chain, embedded in mitochondrial inner membrane
→succinate oxidized, electrons transferred to FAD, producing fumarate and FADH₂
rxn (along with next 2 steps) analogous to 1st three steps of β-oxidation of fatty acids

7th step of TCA: fumarase

reversible

addition of water to double bond

comparable to 2nd step of β-oxidation of fatty acids

8th step of TCA: malate dehydrogenase

reversible

oxidation rxn, producing oxaloacetate, NADH, H⁺

proceeds in forward direction despite very large positive ΔG b/c oxaloacetate is present at such low concentrations and citrate synthase has large negative ΔG

comparable to 3rd step of β-oxidation

Regulation of TCA

logical → high-energy demand stimulates TCA, resting/fed state inhibits

Allosteric regulation and substrate availability are 2 mechanisms that influence rate of TCA on day-to-day basis

cannot increase TCA intermediates through general means such as adding acetyl-COA, specific rxns required

Starving cell (go): low ATP, high ADP/AMP, low NADH, high NAD⁺, low citrate/succinyl CoA, high calcium levels

Satiated cell (stop): high ATP, low ADP/AMP, high NADH, low NAD⁺, high citrate and succinyl-CoA

Regulation of TCA via substrate availability

levels of NAD⁺, oxaloacetate, acetyl-CoA are limiting factors

Regulation of TCA via allosteric regulation

important enzymes: citrate synthase, alpha-ketoglutarate, isocitrate dehydrogenase (also PDC)

ATP inhibits, ADP/AMP stimulates

Pathology associated with TCA

inhibition of PDC will affect every cell
clinical presentations will be associated with most metabolically active tissues: neurons
blocking by limiting thiamin availability (vitamin B₁) causes Wernicke encephalopathy and BeriBeri
→associated with decreased thiamin intake
→Wernicke seen in lots of alcoholics due to poor nutrition, but constant presence of EtOH in CNS doesn't help
PDC deficiency (PDCD) manifests with similar neurological disturbances seen in thiamin deficiency
→patients survive because PDC only links glycolysis, glycolysis can function on its own

Malate-Aspartate cycle

used to oxidize NADH back to NAD⁺, reaches to mytosol where NADH can deal some electrons to OxPhos chain

important in liver, kidney, heart where tissue has steady and constant demand for energy

only reason we do this is b/c inner membrane of mitochondria is highly selective and does not allow oxaloacetate or NADH/NAD⁺ to move freely

conversion b/w malate and oxaloacetate in either direction is mediated by MDH
∙malate→oxaloacetate has high positive ΔG that is only overcome in mytosol due to relative concentrations
∙in cytosol, reverse rxn occurs

relation b/w asparate and oxaloacetate
→Asp aminotransferase mediates shuffling of oxaloacetate into/out of mitochondria

Glycerol-3-P Pathway

for demanding cells (skeletal muscle, neurons) that break down lots of glucose, need faster more efficient way of restoring NAD⁺ levels

can use G3P shuttle to deliver electrons directly from ETC
1. use cytosolic G3P DH to convert DHAP to G3P, oxidizing NADH in process
2. G3P diffuses toward inner mitochondrial membrane and is converted back to DHPA by mitochondrial G3P DH
→during this step electrons delivered directly to coenzyme Q using FADH₂/FAD molecule
→entering ETC at coenzyme Q and skipping proteins 1 and 2 produces less of proton gradient across mitochondrial membrane (2 ATP short as opposed to malate-aspartate shuttle)

Delivery of Fatty Acids to mitochondria

to absorb, must break down TAGs to free fatty acids and glycerol backbone

hydrolytic lipases produced by pancrease are hydrophilic, have tough time reaching hydrophobic fat globules in intestine

gall bladder releases biles salts into small intestine, primarily molecules of cholesterol linked to amino acids, giving them amphipathic qualities, can emulsify fat globules and provide aqueous lipases easy access

once broken down, free fatty acids and glycerol are taken up by small intestine, re-esterified and linked with various apolipoproteins (ApoC II)
→once fat globules assembled they are deemed chylomicrons, which roam around body, dropping off fat wherever apolipoproteins are recognized by receptors on capillary wall until they reach liver
→TAGs must be broken down again to get into some peripheral cells, only to be re-esterified

if liver wants to export fats, does so via VLDL
→deposits fat to extra-hepatic tissues, becomes more dense (LDL)

Mobilizing Fat

in adipocytes, fat stored as TAGs located in globules encased by perilipin
→for hormone sensitive lipase to access TAGs, must get through perilipin coat
→PKA is stimulated to phosphorylate both perilipin and HSL to do so, phosphorylated perilipin will dossociate from globule, activated HSL will acess tag
→→→free FAs released, carried into blood stream by albumin

fat insides adipocytes constantly in flux, cyclical fashion

Free Fatty Acids

Free FAs must be activated with terminal CoA

fatty acyl-CoA synthase responsible for replacing carboxylic head group of FFA with a CoA
→includes priming with AMP before Acyl-CoA added

for activated FFA to gain access to mitcohndria, must be linked with carnitine
→carnitine acyl transferase esterifies FFA with carnitine molecules, gets FFA past outer mitochondrial membrane

β-Oxidation

Fatty acid oxidation that produces NADH, FADH₂, acetyl-CoA
→NADH, FADH₂ donate electrons to ETC

enzymes that help complete will proceed through cycle that generates pair of electrons and acetyl-CoA for every removal of two carbons

Entire process repeats until we have cleaved 16-carbon fatty acid into 8 acetyl-CoA units

Steps of β-Oxidation

1. Acyl-CoA dehydrogenase
2. Enoyl-CoA dehydrogenase
3. β-Hydroxyacyl-CoA dehydrogenase
4. Thiolase (acyl-CoA acetyltransferase)

Step 1 of β-Oxidation: Acyl-CoA dehydrogenase

similar to succinate dehydrogenase of TCA

involves oxidation of alpha and beta carbons

palmitoyl-CoA will be converted to trans-Δ2-enoyl-CoA, takes place on immer mitochondrial membrane where enzyme is located
→enzymes released harnessed by FAD, which ultimately ferries electrons to coenzyme Q

*acyl-CoA dehydrogenase is also analogous to Complex II in ETC*

Step 2 of β-Oxidation: Enoyl-CoA dehydrogenase

similar to fumerase

hydrate double bond just formed b/w alpha and beta carbons
→beta-carbon receives hydroxyl

trans-Δ2-enoyl-CoA → L-β-hydroxyl-acyl-Coa

sets stage to remove acetyl-CoA, first need to convert β-hydroxy to β-keto

Step 3 in β-Oxidation: β-Hydroxyacyl-CoA-dehydrogenase

similar to MDH

oxidized β-hydroxy carbon into keto, allows us to reduce NAD⁺ into NADH

L-β-hydroxy-acyl-CoA → β-Ketoacyl-CoA

Step 4 in β-Oxidation: Thiolase

aka acyl-CoA acetyltransferase

simple nucleophilic attack by sulfur of CoA-SH on β-carbon, creating new acyl-CoA molecule and generating Acetyl-CoA

Path of Electrons from Acyl-CoA dehydrogenase

Acyl-CoA DH is bound to inner mitochondrial membrane

ETF - electron-transferring flavoprotein

Oxidation of Mono-unsaturated Fatty Acids

β-oxidation will proceed as normal until it encounters double bond in Δ³-Δ² position

to continue, but convert cis-double bond and move its position one carbon over
→enzyme Δ³,Δ²-enoyl-CoA isomerase performs this function, allows us to skip acyl-CoA DH

good example: oleic acid

Oxidation of Odd-Numbered Fatty Acid Chains

only made in plants, but must be able to digest them once ingested

only difference from normal β-oxidation is what we do once we get to last three carbons

ex: Propionyl-CoA → need to add another carbon to split resulting 4-chain chain into last 2 acetyl-CoA molecules

2 steps

Step 1 of Oxidation of Odd-Numbered FA chains: Propionyl-CoA carboylase

addition of carboxyl group converting propionyl group to D-methlmalonyl-CoA

necessary cofactors: HCO₃⁻, ATP, Biotin

Step 2 of Oxidation of Odd-Numbered FA chains: methylmalonyl-CoA epimerase & methylmalonyl-CoA mutase

need L, not D-stereoisomer

epimerase converts starting point into L-methylmalonyl-CoA

from here, need to create linear four carbon chain
→function carried out by mutase, forms succinyl-CoA, requires coenzyme B₁₂ to move carboxyl group
→now ready to product 2 acetyl-CoAs

Benefits and Limitations of Fatty Acid Oxidation

breakdown of FAs into acetyl-CoA units cannot help generate TCA cycle intermediates
→one exceptionL propionyl-CoA conversion to succinyl-CoA
→small amount of succinyl-CoA produced during breakdown of odd-numbered FAs can contribute to generation of TCA cycle intermediates

FAs are incredibly great stores of energy
→1 palmitate molecule (16C) can result in 108 ATPS
→hydrophobic, can be stored in great quantities

Ketone Bodies

produced in liver cell mitochondria when we have excess of acetyl-CoA, which only occurs when we have excess β-oxidation

acetoacetate and D-β-hydroxybutyrate are ketone bodies that serve as sources of fuel for extra-hepatic tissues, usually during periods of starvation
→brain can use ketone bodies for 33% of energy needs during starvation, reducing need for glucose

acetone also ketone body but not for fuel, produced in small amounts, exhaled

will be released by liver, distributed to various extra-hepatic tissues that have cells with mitochondria capable of converting them back to acetyl-CoA units
→acetoacetate may also be converted to acetone via an enzyme
→rxn may also occur via spontaneous decarboxylation w/o enzyme
→acetone produced because of dangerous excess of acetyl-CoA and other ketone bodies, not as fuel, wants to be exhaled to detox blood

Ketogenesis

synthesized from acetyl-CoA produced by β-oxidation

begins with condensation of 2 acetyl-CoA catalyzed by thiolase (reversal of last step in β-oxidation)

3 steps

Step 1 of Ketogenesis: Thiolase

reverse of last step in β-oxidation

combine 2 acetyl-CoA units to produce acetyoacetyl-CoA

one CoA-SH released too

Step 2 of Ketogenesis: HMG-CoA synthase

condensation rxn combining acetoacetyl-CoA with another acetyl-CoA

rxn requires water, releases another CoA-SH group

product is β-hydroxy-β-methylglutaryl-Coa (aka HMG-CoA)
→also produced during cholesterol synthesis but happens in smooth ER, in ketogenesis forms in mitochondrial matrix

Step 3 of Ketogenesis: HMG-CoA LYase

HMG-CoA lyase cleaves acetyl-CoA unit producing acetoacetate
→acetyl-CoA added in previous step just as primer for this step

Formation of D-β-hydroxybutyrate

acetoacetate is 1st ketone body, but predominant form in blood is reduced version, D-β-hydroxybutyrate
→conversion carried out by enzyme D-β-hydroxybutyrate dehydrogenase, requires oxidation of NADH

Ketone Body Breakdown

once these emergency energy compounds ahave been distributed to mitochondria of extra-hepatic tissues, two steps needed to convert them back to acetyl-CoA
→forst, any D-β-hydroxybutrate must be oxidized back to acetoacetate by D-β-hydroxybutyrate dehydrogenase, requires NAD⁺

under starvation conditions, FAs are broken down to acetyl-CoA units, but w/o glucose we cannot generate TCA cycle intermediates needed to metabolize excess acetyl-CoA → means we can't actually accelerate rates of acetyl-CoA use and therefore our liver converts them to ketone bodies

Step 1 of Ketone Body Breakdown: β-ketoacyl-CoA transferase

converts ketone body into acetoacetyl-CoA

uses succinyl-CoA to attach CoA-S- to product

β-ketoacyl-CoA transferase not present in brain under normal conditions but is activated during starvation to allow use of ketone bodies

not catalyzed in liver

Step 2 of ketone body breakdown: Thiolase

thiolase splits 4-carbon compounds into 2 acetyl-CoA units

also adds another CoA-S

Fatty Acid Synthesis

site of synthesis: cytoplasm

starting material for synthesis: acetyl-CoA

source of acetyl-CoA units: mitochondria

problem: acetyl-CoA cannot be transported across mitochondrial inner membrane

Citrate shuttle

site of FA metabolism is cytoplasm but starting compound for FA synthesis is acetyl-CoA, which comes from mitochondria

acetyl-CoA cannot cross inner mitochondrial membrane, so attached to carrier → citrate

acetyl-CoA units in matrix attached to oxaloacetate by enzyme citrate synthase (from TCA), citrate can then move through transporter and into cytosol

in cytosol, citrate lyase frees acetyl-CoA unit in energy-dependent fashion using ATP

REMEMBER - CoA-SH removed by synthase, added by lyase

how do we get NADPH here:
→oxaloacetate converted to malate via MDH and then malate is decarboxylated to pyruvate by malic enzyme
→malic enzyme delivers reducing equivalents to NADP⁺ forming NADPH (also produced in PPP)

Step 1 in FA synthesis: Acetyl-CoA carboxylase

Acetyl-CoA carboxylase enzyme will until produce malonyl-CoA, which inhibits CAT1

know that we are using bicarbonate to attach carboxyl group to an acetyl-CoA
→often utilizes biotin as cofactor
→specifics not important

Step 2 in FA synthesis: Fatty Acid Synthase

conglomerate of enzymes in one polypeptide that helps assemble FAs

acyl carrier protein (ACP) is in center w/position that can attach malonyl-CoA
→before attached, want to attach acetyl-CoA to KS subunit of FA synthase

Condensation: once synthase charged with malonyl and acetyl group, have nucleophilic attack by malonyl group on acetyl group and decarboxylation

Reduction: now 4-carbon chain attached to ACP, NADPH used to reduce β-keto group (opposite of β-oxidation)

Dehydration: loss of water to form enoyl group

Reduction: NADPH used to reduce enoyl group to acyl group, results in acyl chain lengthened by 2 carbons, longer chain then transferred to KS subunit and process starts over again

Storage lipids

TAGs

neutral

Membrane lipids

Phospholipids
→glycerophospholipids
→sphingolipids

Glycolipids
→Sphingolipids

POLAR

Glycerophospholipids

Glycerol backbone

2 FA side chains

Phosphoester that can be further modified with different groups

Ester-linked glycerophospholipids

Plasmalogen & Platelet-activating factor

Plasmalogen

ester-linked glycerophospholipid
→ether-linked alkene at position 1, resistant to phospholipases

don't know what function is

abundant in cardiac tissue

most common group is choline

elevated levels seen in metastatic cancers (possible biomarker?)

Platelet-activating factor

ester-linked glycerophospholipid
→ether-linked alkyl group at position 1 AND acetyl ester at position 2

slightly more soluble than plasmalogen → able to circulate in blood as molecular signal

when bound to receptors, triggers events → mediates release of granules from platelets, i.e. serotonin → causes platelet aggregation

inactivated by cleavage of acetyl ester

Sphingolipids

sphingosine backbone

FA at position 2

various groups at position 3

Glycerophospholipid Synthesis

Glycerol-3-phosphate precursor for TAGs and glycerophospholipids
→synthesis of TAGs and glycerophospholipids share common origin

Phospholipids are result of condensation of phosphoric acid with 2 alcohols
→NOT SIMPLE MECHANISM IN PICTURE

Two strategies for head group attachment: activated diacylglycerol & activated head groups

Eukaryotic synthesis of anionic phospholipids PG, cardiolipin and PI is similar to that of bacteria via CDP-diacylglycerol

Activated diacylglycerol vs. Activated head group in glycerophospholipid synthesis

Activated diacylglycerol:
∙energy activated form of diacylglycerol with CDP, can react with head group
∙bacteria employ this strategy
→can be anionic, zwitterionic, pathway changes

Activated head group:
∙energy activated hydroxyl group on head group with CDP to react with diacylglycerol

Respiration

involves expansion and contraction of lung alveoli

lungs have surfactant on alveoli
→difficult to separate alveoli w/o surfatant → force must be greater to overcome surface tension
→surfactant made up of mainly DPPC (Dipalmitoylphosphatidylcholine) and unsaturated PC (phostphatidylcholine), also have phosphatidylglycerol, cholesterol, FAs, proteins and other lipids

Fetal Lung Surfactant Production

L/S ratio is usually >2.0

in premature birth, 40% chance of RDS (L/S ratio is 1.5-1.9)

if L/S <1.5, 70% RDS

Sphingolipid biosynthesis

4 stages:
∙synthesis of sphinganine, an 18-carbon amino alcohol
∙attachment of fatty acid via amide bond
∙desaturation of alkane chain of acylsphinganine (results in ceramide)
∙head group attachment
→picture deceiving, because phosphocholine is attached here, not just head group

previously thought that sphingomyelin was synthesized from N-acylsphingosine and CDP-choline, now known to be minor pathway

most prevalent acyl groups in SM are 16:0 and 18:0

synthesis occurs in Golgi, unlike ceramide and other phospholipids that are synthesized in sER

Cerebroside Biosynthesis

begins with ceramide, one sugar added

glucocerebroside - minor lipid in non-neural tissue, however, is precursor of golobosides and gangliosides

galactocerebroside - common lipid in brain, often referred to as cerebroside

both are linked by β-linkage at position 1 of hexose

Sulfatide Biosynthesis

sulfatides = 15% of brain white matter lipids

PAPS (3'-phosphoadenosine-5'-phosphosulfate) serves as source for activated sulfate group

sulfate transferred almost exclusively to 3-OH of galactose

w/ addition of sulfate, monosaccharide has negative charge, so sulfatides are not neutral sphingolipids

Ganglioside and Globoside Biosynthesis

addition of more sugars that cerebrosides

additions carried out by series of glycosyl transferases (in Golgi)

addition of 2nd NANA to Gm₃ leads to Gd series

addition of GlcNAc to lacosyl ceramide bore NANA leads to Ga₂

overall, more than 60 known gangliosides

when one or more sugars added are sialic acid, generating gangliosides
→large, diverse class of sphingolipids
→allows different sphingolipids to serve as molecular tags or markers

Simple Ganglioside Nomenclature

G = ganglioside
→M = 1 sialic acid
→D = 2
→T = 3
→Q = 4
→also have other letters based on branching

# = 5- moieties (excluding SA)

Regulation of Sphingolipid Synthesis & Degradation

carefully controlled

normally, constant turnover of sphingolipids, which occurs in lysosomes

most often in glycolipid storage diseases, biosynthetic rate remains normal but mutation exists in structural gene for hydrolase
∙impaired degradation of sphingolipids results in accumulation of membrane lipids = severe clincal mutations
→accumulation of membrane lipids in lysosome damages lysosome functions
∙must be able to degrade sequentially just as synthesized

Lipid storage diseases

Generalized gangliosidosis

Tay-Sachs disease

Sandhoff's disease

Fabry's disease

Gaucher's disease

Niemann-Pick disease

Symptoms of lipid storage diseases

hepatosplenomegaly
→enlargement of liver, spleen

scoliosis

corneal opacity
→classic sign

macular spots

ataxia, dystonia, hypotonia
→due to effect on neuronal tissue

macroglossia

Lipid Functions

vast bulk of cellular lipids serve as bilayer structural components

can serve as modulators of activity of both soluble and membrane-bound proteins
→especially acidic phospholipids

phosphatidylionsitol serves as part of tether of GPI-anchored membrane proteins

ether-linked glycerophospholipids, such as platelet-activating factor, can act as molecular signals

derivatives of phosphatidylinositol act as intracellular second messengers in signal transduction pathways involved with many aspects of cellular regulation

FA metabolites, the eicosanoids, act as potent, short-lived hormone-like molecules

Phosphatidylinosital derivatives serve as intracellular signals

generates diacylglycerol, which activates protein kinase C, leads to regulation of other enzymes by protein phosphorylation

head group leads to release of intracellular calcium, leads to regulation of other enzymes by calcium

Eicosanoids are derived from arachidonic acid released from phospholipids

phospholipids → arachidonate → prostaglandins, thromboxanes, leukotrienes, lipoxins, hepoxilins, epi-llipoxins

arachidonic acid has 4 unsaturated bonds on it
→gives rises to 2 and 4 series
→if it had 3 unsaturated bonds, it would give rise to 1 and 3 series
→5 unsaturated bonds → 3 and 5 series

Linoleate & α-linoleate are essential dietary fatty acids for mammals

elongation steps, carried out in sER and mitochondria, involve addition of acetyl groups from coenzyme A

only plants have ability to desaturate linoleate to α-linoleate, humans desaturate to γ-linoleate

α-linoleate vs. γ-linoleate

α-linoleate is ω-3 fatty acid
→has unsaturated bond that is only 3 positions in from ω-carbon
→flax seed, plants, seafood (animals that eat lots of it) are rich in it

γ-linoleate is ω-6 fatty acid
→has unsaturated bond that is 6 positions in from ω-carbon

study showed that ω-3 FA reduces risk of sudden cardiac death, also lowers TAG levels

Desaturation of palmitate and stearate

Desaturation of palmitate (16:0) and stearate (18:0) is carried out by fatty acyl-CoA desaturase is mixed-function oxidase, w/ both acyl chain and NADPH being oxidized

Plant acyl desaturases

Located in ER, convert oleate (by far most often at sn₂ position) of PC into linoleate and linolenate w/ sequential introduction of unsaturated bonds at carbons 12 and 15
desaturases, which may also act upon free FAs, are located in chloroplasts

Phospholipases cleave glycerophospholipids at different sites

named for where they cleave

See More

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