Biochem Exam III Review.2

BIOCHEMISTRY

Lecture 21. MITOCHONDRIA.

Mitochondrial Structure and Composition.1. Outer membrane

a. Permeable to small metabolites b. Does not serve as a barrier to proton diffusion

2. Inner membranea. Contains key proteins for energy production (e.g. ATP synthase) and transport systems for necessary

metabolites b. High proportion of cardiolipin (diphosphatidyl glycerol) which enhances its lack of permeability to

protons3. Human mitochondrial DNA

a. Small, circular maternally-derived DNA (~17 kb)b. Endosymbiotic origin (own system for DNA replication and transcription)

4. mtDNA differs from nuclear DNAa. Contains very little intronsb. Genetic code differs also; encodes 37 genes:

i. 2 structural ribosomal RNAs ii. 22 transfer RNAs needed for mitochondrial protein synthesis

iii. 13 of the 70 proteins that form the ETC machinery

5. Majority of the hundreds of proteins in the mitochondria are imported from cytosol into the mitochondria; energy dependent

a. Presequence (matrix targeting sequence): sequence at the N-terminus that directs precursor proteins to the mitochondria

b. Two paths into the matrixi. Translocases of the outer membrane (TOMs)

ii. Translocases of the inner membrane (TIMs)

Mitochondrial Respiratory Chain - Structure & Function1. Substrates for respiratory chain:

a. NADH (3 ATP generated)b. Complex I (NADH dehydrogenase)c. FADH2 (2 ATP generated)d. Complex II (succinate, acetyl-CoA,

glycerol, phosphate dehydrogenases)e. Prosthetic groups: harbor the

transferable electrons i. Pyridine Nucleotides (NAD+)

1. NAD+ accepts two electrons and a proton to generate NADH

2. Remaining proton enters the aqueous medium of the cell

ii. Flavoproteins1. Complex II : FAD

undergoes reversible oxidation and reductiona. FAD-linked proteins:

i. Succinate dehydrogenase; glycerol-3-phosphate dehydrogenase; Acetyl-CoA dehydrogenase

2. Complex I : NADH dehydrogenase enzyme accepts electrons

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from NADH and utilizes FMN (flavin mononucleotide)3. The flavoproteins usually have a more positive reduction potential than

NAD+/NADH, making electrons more readily transferred to flavoproteins from NADH, regenerating NAD+

iii. Coenzyme Q (Ubiquinone)1. Transfers electrons from Complexes I and II to complex III2. Benzoquinone derivative inserted into inner membrane3. Fully oxidized: CoQ4. Two electron reduced form: CoQH2

iv. Cytochromes 1. Proteins containing the prosthetic group heme2. Transfer electrons sequentially from CoQH2 to molecular

oxygen3. Cytochrome b : component of complex III

a. has the most negative reduction potential; accepts electrons from CoQH2; the protons removed from CoQH2 during this process are released and transported from the mitochondrial matrix into the intermembrane space

4. Cytochrome c1 : also component of complex III; transfers electrons to cyto c

5. Cytochrome c : on outer side of the inner membrane (facing the intermembrane space)

6. Cytochrome Oxidase : contains two hemes: Cytochrome a and Cytochrome a3 in addition to copper

a. Accepts 4 electrons from cytochrome c and transfers them via cytochrome a3 to a molecule of oxygen

b. As it picks up 4 protons from the medium, it generates two molecules of water

v. Iron-Sulfur proteins (Fe-S*) 1. Proteins containing non-heme iron prosthetic groups2. Serve as electron carriers 3. Components of Complexs I, II, and III

2. Complex I a. NADH: CoQ reductase

3. Complex II a. Succinate: CoQ reductase

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4. Complex III a. Reduced CoQ: cyt c

reductase5. Complex IV

a. Cytochrome oxidase6. Electron transfer by

respiratory chain is coupled (physically, energetically) to the transfer of proteins

7. Total of 10 protons pumped from matrixa. 4 protons during transfer of 2

electrons through Complexes I and III

b. 2 protons during transfer of 2 electrons through Complex IV

c. Complex II is not a proton pump

8. Proton-motive forcea. pH gradient & membrane potential (DYm): source of potential energy for

ATP synthesis9. The addition of an inhibitor together with a substrate will cause all components

proximal to the block to be fully reduced and components distal to the block to be fully oxidized

a. Complex Ii. Rotenone (insecticide) binds to Complex I and prevents the reduction

of ubiquinone; also Amytal and Piercidin Aii. NADH cannot be used but Electron flow can continue because the

oxidation of succinate at Complex II allows electrons to enter the chainb. Complex III

i. Antimycin inhibits electron transfer through CIII c. Complex IV

i. Cyanide and azide bind tightly to the oxidized (ferric) form of cytochrome a3

ii. Mitochondrial respiration and energy production ceases; cell death rapidly occurs

iii. Carbon monoxide also inhibits CIV, but by binding to the reduced (ferrous) form of cytochrome a3

Transport Systems to Move Biomolecules into and out of the Mitochondrion

1. Sources of substrates (reducing equivalents) for the ETCa. Glycolysis: NADH generated in cytosol

i. Pyridine nucleotides cannot penetrate the mitochondrial inner membraneb. Amino Acid Oxidation: generates pyruvate or acetyl-CoA, which can be further oxidized to CO2,

generating NADHc. Fatty Acid Oxidation: generates NADH, reduced flavoprotein, and acetyl-CoA

i. NADH: enters ETCii. Flavoproteins: reduces ubiquinone

d. TCA cycle

i. Four of the oxidative reactions of the TCA cycle, that are catalyzed by dehydrogenases, generate NADH; the reaction catalyzed by succinate dehydrogenase transfers electrons directly to ubiquinone

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2. Malate-Asparate Shuttle a. Malate can serve as an electron carrier between the cytoplasm and

mitochondriab. Depends on presence of

i. Two enzymes:1. Malate dehydrogenase (MDH)2. Glutamate-oxaloacetate transaminase (GOT)

ii. And two transporters:1. α-Ketoglutarate transporter2. Glutamate-asparate transporter

c. Cytoplasm: NADH is used to reduce OAA to malatei. Enters mitochondria via α-Ketoglutarate transporter

d. Mitochondria: Malate reoxidized to OAA, regenerating NADH (to be used for oxidative phosphorylation)

i. OAA is used to form glutamate by GOT, generate asparate which is transported back to cytoplasm

3. Glyerol Phosphate Shuttle a. Cytoplasmic NADH is used to reduce dihydroxyacetone phosphate

(DHAP) to glycerol-3-phosphate (G-3-P)b. G3P is reoxidized by a FAD-linked glycerol phosphate

dehydrogenase present on surface of mitochondrial inner membrane, which transfers electrons to ubiquinone

c. Unlike malate-asparate shuttle, no compounds, only electrons, are transferred through mitochondrial inner membrane

d. Since the electrons from mitochondrial FADH2

feed into the oxidative phosphorylation pathway at coenzyme Q (as opposed to NADH-ubiquinone oxidoreductase [complex I]) only 2 moles of ATP will be generated from glycolysis rather than 3

1. ATP Synthase (a.k.a. ATPase or Complex V)a. Dissipates the proton gradient, using the energy to synthesize ATPb. Inhibitor of ATP synthase: Oligomycin

c. Complex consists of two components with different functionsi. F0 component: forms transmembrane channelii. F1 component: contains 3 identical active sites in which synthesis of ATP from ADP and Pi

occurs1. The conformational changes cause the dissociation of the product ATP and

promotes the ADP phosphorylation reactiond. Active center of ATP synthase is accessible only from the matrix side of the inner membrane

i. ADP and Pi have to enter the mitochondria, and ATP has to leave it; all via two transporters:

1. Pi/OH- exchanger (Pi in; OH- out of matrix and into cytosol)2. ATP/ADP exchanger (inhibited by atractyloside)

2. In the mitochondrial OXPHOS system electrons flow to complexes of a lower reduction potential to an oxidized molecule of higher reduction potential

3. In “coupled” mitochondria change in the rate of respiration leads to the change in the rate of ATP synthesis and vice versa

a. Respiratory control ratio (RCR): ratio of the rates of both respiration and oxidative phosphorylation, with and without ADP, including how tightly couples the mitochondria are

4. Uncouplers disrupt the interdependence of respiration and ATP synthesis by allowing protons to bypass the ATP synthase (dissipate the H+ gradient)

a. Causes respiration to proceed at maximal rate, no longer constrained by the increase in membrane potential; The energy goes to heat

b. Uncouplers: FCC; dinitrophenol; termogenin or UCP1 (brown fat- burning calories to generate heat)

c. If the ETC is interrupted at a step after formation of the electrochemical

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proton gradient, an uncoupler will restore respiration but not ATP synthesis

d. Possible causes for this situation:i. Lack of ADP or Pi

ii. Inhibition of ADP/ATP translocator (e.g. by atractyloside)iii. Inhibition of phosphate transporteriv. Inhibition of ATP synthase (e.g. by oligomycin)

5. Uncouplers will not restore respiration if the ETC itself is inhibited (e.g. by cyanide) or if there is a lack of respiratory substrates within the matric (e.g. steps before the formation of the proton-motive force)

Mitochondria and human disease1. Mitochondrial Permeability Transition (MPT) is the loss of the inner

mitochondrial membrane impermeability to solutes caused by extended opening of the Permeability Transition Pore (PTP)

a. No longer a barrier against protons re-entering matrixb. Leads to the cessation of ATP synthesis and swelling of the mitochondrial matrix

apoptosis and necrosisc. CypD-deficient Mice are Resistant to Ischemia/Reperfusion-induced Cardiac Injury

2. Reactive Oxygen Species a. Incorrect or overactive functioning of the respiratory chain (e.g. in presence of

inhibitors or when excess NADH is present) leads to an increase in ROS synthesis

b. ROS can damage DNA, fatty acids, triglycerides and proteinsi. Detoxification of ROS occurs in pentose phosphate pathway

c. Are implicated in many pathological processes such as diabetes, Alzheimer’s, Parkinson’s, and aging

Lecture 21b. Mitochondrial Myopathies.

Mitochondrial Myopathies1. Mitochondrial Disease

a. Multi-system involvementb. Progressive and variable coursec. Easy fatigabilityd. Unusually severe reactions to infectious illnessese. Autonomic symptomsf. Family history of similar symptomsg. Even dysmorphic features

2. Mitochondrial Myopathies can result from defects in nuclear DNA or in mitochondrial DNAa. Majority of mitochondrial proteins are coded for by nuclear DNA, translated in the cytosol and imported

into the mitochondrion; allows the possibility to have a mitochondrial myopathy inherited from the father

3. Defects in mitochondrial genome (Maternal inheritance)a. Account for majority of the disorders

4. Each cell contains hundreds of mitochondria, thus thousands of copies of mitochondrial DNA

a. Heteroplasmy : cell may contain populations of mutant and of normal mitochondrial DNAs, which may not be distributed equally to daughter cells after meiosis and mitosis. This would allow the mitochondrial DNA

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populations to become successively more homogeneous (Homoplasmic), yielding cells with predominantly normal or predominantly mutant mitochondrial DNA

5. Threshold effect : severity of symptoms is a combination of the degree of impairment of function caused by the mutation, the proportion of mutant mitochondrial DNA, and the dependence of the particular tissue on oxidative phosphorylation

6. Leber’s Hereditary Optic Neuropathy (LHON) a. Acute adult onset blindness with rapid loss of visionb. Maternal mode of inheritance; males affected more than femalesc. Due to missense mutation causing a single amino acid substitution in Complex I peptide

7. Kearns-Sayre Syndrome (KSS)a. Ocular myopathy; retinitis pigmentosa and lactic acidosisb. Single mtDNA deletion, but size and position of deletion differs among patientsc. Rate of replication of mtDNA is proportional to the molecule’s length

i. The shorter, deleted molecules have a replicative advantage over the longer, normal molecules leads to an increasing proportion of mtDNA molecules with the deletion

d. Symptoms show age-related progression 8. Myoclonus Epilepsy with Ragged Red Fibers (MERF)

a. Ragged red muscle fibers and abnormal mitochondriab. Point mutation in a loop of mitochondrial tRNA, causing inhibition of mitochondrial protein synthesis;

has a larger effect on Complexes I and IV9. Diagnosis:

a. The only way to be 100% sure of the diagnosis is to identify a DNA mutationb. Biochemical features: Abnormalities in blood, urine, spinal fluidc. Radiologic features: MRI scan; MR spectroscopyd. Physiologic testing: resting energy expenditure; exercise testinge. Tissue biopsy: Abnormalities in size, shape, or number of the mitochondria, or their structuref. “Organ inventory”: Testing for multiple organ dysfunction:

10. Determination:a. Experiment 1: Is the problem with a protein coded for by N-DNA or Mt-DNA?

i. Fuse cells from:1. Patient (with destroyed mtDNA) and2. Normal individual (with nucleus removed)

ii. if mitochondria are normal, problem resulted from a defect in the patient’s mitochondrial DNA

iii. if the mitochondria are not normal, the problem resulted from a defect in the patients nuclear DNA

b. Experiment 2: Reverse situation11. Enzyme activity

a. Can assay each segment of the ETC separately by selective use of inhibitors and artificial electron donors and acceptors

b. Can also determine whether mitochondria are tightly coupled by measuring respiratory control ratio 12. Level of components

a. Determination of CoQ levels or presence or absence of different peptides 13. Determine whether mitochondrial function has been compromised by an external agent

a. Promote energy productionb. Reduce energy loss

14. Procedure/ Treatmenta. KSS treatment: giving the patient the cofactors (such as coQ) and oxidizable substrates to keep the

residual ETC functioning at its full capacityb. To circumvent defects in Complexes I and II: use electron donors to reduce Complex III or IV directlyc. Fusing the DNA sequence that would encode the normal protein in the cytosol to a sequence that would

encode a matrix targeting sequence at the N-terminus of a protein

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Lecture 22. HEME

1. Porphyrins are important prosthetic groups of many proteins; all have the same porphin ring; sidechains on pyrrole rings distinguish prophyrins

a. Type III : physiologic form of porphyrins (ring D)b. A porphyrinogen spontaneously oxidizes to a porphyrin

i. porphyrinOGENS (reduced porphyrins) are intermediates in heme biosynthesis

2. All cells synthesize heme; Most is synthesized in bone marrow (presence of RBCs); rest in liver

3. Heme biosynthesis:a. SuccinylCoA and Glycine are combined by δ-

aminolevulinaste synthase (ALAS) to form a delta-aminolevulinate

i. Occurs in the mitochondria; product is transported into cytoplasm

ii. Slowest reaction; key (only) regulated stepb. δ -ALA dehydratase condenses two molecules of

δ-ALA to form porphobilinogeni. Enzyme is a target for lead poisoning,

where lead displaces the zinc atomsc. Condensation of four molecules of porphobilinogen to form

uroporphyrinogen, with the loss of 4 molecules of ammoniai. Enzyme: Porphobilinogen deaminase (uroporphyrinogen synthase)ii. Uroporphyrinogen co-synthase assures closure in type III form

1. Without co-synthase, make type I which can be converted to coproporphyrinogen but not protoporphyrinogen; leads to deposits in skin & photosensitivity

d. Decarboxylation forms Coproporphyrinogeni. Molecule now more hydrophobic; diffuses back into mitochondria

e. Decarboxylation and oxidation forms Protoporphyrinogenf. Protoporphyrinogen is rapidly oxidized to form protoporphyring. Protoporphyrin captures iron to form Heme

i. Catalyzed by Ferrochelatase

4. Liver , and other non-erthroid cells

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a. Can synthesize heme for cytochromes, particularly cytochrome P450 enzymes, which metabolize drugs and other endogenous intermediates

b. δ-amino levulinaste synthase (ALAS) is regulated by free hemec. Accumulation of heme causes :

i. Inhibition of gene transcription, 1. Inhibition of synthesis of ALAS1 :

a. Decreased mRNA stability (heme)b. Decreased transcription of ALAS1 gene (insulin)c. Steroid hormones increase ALAS1 synthesis

ii. Inhibition of transport of newly synthesized ALAS1 enzyme into mitochondria

iii. Allosteric inhibition of ALAS1 by heme in mitochondriad. Drugs upregulating CYPP450 ( barbiturates ) increase heme synthesis

i. Any enzyme that uses heme will conjugate to it and decrease the level of free heme in mitochondria and thus increase ALAS1

5. Erythroid cellsa. Heme is synthesized in immature erythrocytes b. Mature erythrocytes do not contain mitochondria and do not synthesize heme.

c. Express a gene for ALAS-2 (different from liver)d. Regulation:

i. Iron 1. Increases translation of ALAS2 (displaces inhibitor from

mRNA) 2. Increases ferrochelatase activity

ii. Erythrocyte-specific transcription factors act on ALAS2 gene promoteriii. Inhibition of ALAS2 transport into mitochondria (heme feedback

inhibition) 6. None of the intermediates of heme biosynthesis accumulate because after the first

step, all the steps are extremely fast; also have negative feedback control7. Porphyrias : diseases associated with defects in heme synthesis

a. Levels of free heme decrease, which causes the first step’s activity to increase (partial release from feedback inhibition), resulting in an even larger increase in amount of intermediates up to the defect

b. Porphyrias are associated with abnormally high levels of δ-amino levulinaste acid synthase activity and an overproduction of the intermediates up to the step at which the deficiency occurs

c. Therapies try to decrease activity of the first step: give hemin; glucose

d. e.g. patients cannot use any drugs that will interact CYPP450 or steroids

e. Diagnosis: i. Urine PBG test

ii. Analysis of excreted (ALA,iii. PBG, Uroporphyrinogen – urine;iv. Uroporphyrinogen, Coproporphyrinogen, Protoporphyrinogen –

stool sample)

8. Steps in heme degradation

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a. A cytochrome P450 mixed function oxidase converts heme to biliverdin (a green compound)

b. Biliverdin reduced to yellow-brown compound bilirubinc. Free bilirubin is insoluble; bilirubin is bound to serum albumin for transport to

the liveri. Bilirubin is very insoluble & forms deposits; takes a while for

macrophages to get rid of it (e.g. extended presence of bruise)d. Bilirubin is conjugated to glucuronic acid in the liver

i. Conjugated bilirubin is water-soluble and is excreted into bileii. Bile is collected into the gallbladder, and drains via the common bile

duct into the small intestinee. Intestinal bacteria metabolize bilirubin to fecal and urinary pigments

9. Liver disease : accumulation of bilirubin10. Excessive hemolysis : accumulation of bilirubin11. Occlusion of bile duct : accumulation of conjugated bilirubin 12. Jaundice in newborns due to defects in heme breakdown

a. Bilirubin can accumulate in fenestrae of the brain if not properly excretedb. Light therapy can help remove bilirubin; isomerizes the h-bonds and breaks them; molecule becomes a

lot more hydrophilic, dissolves, and is easily excreted

LECTURE 23: INTRODUCTION TO LIPID METABOLISM

1. Lipid: a. Isoprenoids

i. Membranes, signals, cofactors, fat soluble vitaminsb. Fatty acid bases

i. Energy storage, fat deposits (fat is the most efficient way of storing energy), membranes, signals

2. Fatty acids a. Numbering: carboxyl carbon =1b. FA symbol: # before colon = total length of

chain (how many carbons); number after colon = how many double bonds; Superscript = list of double bond positions with regard to carboxyl group

c. Unsaturated have double bondsi. Saturated: Stearic Acid 18:0

ii. Monounsaturated: Oleic Acid 18:1Δ9

iii. Polyunsaturated Fatty Acid: aLinolenic Acid 18:3 Δ 9,12,15

3. Triacylglycerols (TAGs) = dietary and storage fatsa. Fully hydrophobicb. Formed by dehydration between 3 FAs and glycerolc. Ester linkage (carbon through oxygen to carbonyl carbon of FA)d. Glycerol is symmetric (at the 1 & 3 position)e. Melting point of pure triacylglycerols or mixtures of TAGs (e.g. liquid vegetable oils versus solid

animal fats)

i. Melting points of uncharged FAs (R-COOH) increase with chain length

4. Almost all naturally occurring double bonds are cis; forms a kink in the double bond and decrease in melting point

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a. Can convert cis to trans (=processed foods); Melting point is almost the same as the unsaturated molecule

b. Saturated FA (or trans-unsaturated FA) = high MPc. As number of double bonds increase, melting point decreases (solid liquid)

5. Digestion : 6. Emulsification increases fats’ accessibility to digestive enzymes

a. Bile salts & phospholipids are good emulsifiers

7. Hydrolysis of TAGs by Pancreatic Lipasea. Overall reaction: TAG 2x FA + 2-MAGb. Pancreatic lipase doesn’t recognize olestra (FAs in ester linkages to

hydroxyl groups on sucrose)

8. Absorption :a. To enter intestinal epithelial FAs must be monomers

i. FA salt solubility decreases as alkyl chain length increases

ii. Short to Medium Chain FA Salts (6-12C): Diffuse easily (simple diffusion)

iii. Long Chain FA Salts (14C or longer): Most dietary FAs. Solubility too low to facilitate diffusion into cells

b. Micelles: self-association of amphipathic molecules in water; facilitate absorption

i. Form at the Critical Micelle Concentration (CMC)

c. FA activation & reformation of TAGs drives absorption

i. Used to keep fatty acids inside the cellii. Inorganic Pyrophosphatase Rapidly and

irreversibly converts the covalently linked phosphates to free phosphates

iii. Linking the low ΔG of fatty acyl CoA synthetase to the high ΔG of inorganic Pyrophosphatase makes the reaction irreversible so it doesn’t go backwards in the cell

9. Intestinal Cell Resynthesis of TAGsa. Activated FA can’t diffuse out of epithelial cellb. Transport to body starts with TAG re-synthesisc. Enzymes:

i. Fatty acyl-CoA synthetase ii. Triacylglycerol synthase

10. Digestion Disorders

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11. Similar story for dietary cholesterola. Hydrolysis of cholesterol esters (CEs) to allow absorptionb. Resynthesis of CEs to drive absorption & prepare for transportc. Plant sterols, sitosterols, cannot be used and are actively pumped out of epithelia into intestinal lumen

12. Chylomicrons

LECTURE 24: LIPID TRANSPORT AND FATTY ACID CATABOLISM

1. Very Low Density Lipoprotein (VLDL)a. Very similar to Chylomicrons (but made in the liver)b. Characteristic Apoprotein: ApoB100 not ApoB48

i. ApoB100 is derived from same gene as ApoB48 by RNA editingc. ApoC and E are transferred to it from HDL after release from hepatocytes

2. Intermediate Density & Low Density Lipoproteins (IDL & LDL)a. When cells need cholesterol they will express the LDL receptor on their

surfaceb. Internalization into an endosome, fusion with a lysosome, release of contents

3. LDL as the “bad cholesterol”

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a. LDL, if not taken up rapidly, becomes oxidized and initiates a cascading inflammatory reactionb. Oxidized B100 no longer functions and LDL can no longer be taken upc. Modified LDL stimulates endothelial cells to recruit Monocytes and promotes

differentiation into Macrophages which release Cytokinesd. Macrophages ingest modified LDL, become Foam Cells & begin to diee. Dying foam cells form fatty streak & release metalloproteinases & growth factors that promote smooth

muscle cell growth

4. HDL as the “good cholesterol” a. Can protect against CHD by promoting efflux of excess cholesterolb. HDL is anti-inflammatory; protects against the development of atherosclerosisc. Efflux of cholesterol from foam cells leads to a reduction in foam cell formation

d. How HDL works i. Released as an “empty shell” by the liver & intestine (contains only membrane components &

Apoproteins A, C and E).ii. Transfers Apos C-II & E to other lipoproteins

iii. Removes excess cholesterol from cell surfaces (via serum Lecithin Cholesterol Acyl Transferase (LCAT)) and packs into hydrophobic core

iv. Binds to Scavenger Receptor-BI (HDL receptor) on liver & adrenals for endocytosis & degradation

e. Lecithin cholesterol acyl transferase (LCAT) i. LCAT needs an activated FA to add to membrane cholesterol to turn it

into a cholesterol esterii. Lectin + Cholesterol Lysolecithin + Cholesterol Ester

1. Takes a FA from a membrane phospholipid; converts it into something that can act as a detergent

iii. ApoA-I on HDL stimulates this process

f. Cholesterol Ester Transfer Protein : HDL can also pick up triglycerols by exchanging them for cholesterol esters with chylomicrons, VLDL, and IDL

5. Characterizing lipoproteinsa. Lipoproteins with a small volume (HDL) have more

protein versus lipid high densityb. Large complexes slowed by electrophoresis gel matrix

6. Heart Disease a. Saturated fat increases heart disease risk, but

trans fats are worsei. Trans fat does increase LDL, but

more importantly, lowers HDL7. Genetic Defects in Lipid Transport

a. Hyperlipoproteinemias: Overproduction of one or more lipoprotein

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Condition Frequency Lipoprot. Increased

Causes

Familial Hypercholesterolemia

1:500 LDL (VLDL)

Defective LDL-R

Fam. Combined hyperlipidemia

1:500 LDL ApoB100 overproduction

Fam. Ligand-defective apoB100

1:1000 LDL ApoB binds poorly to R

8. Fatty Acid Catabolism a. Most dietary fat (from chylomicrons) and synthesized fat

(from VLDL) is stored in adipose cells b. Serum albumin allows free FAs to be transported in the

blood without allowing them to act as detergentsc. Acyl-CoA activation keeps FA in the cell

d. Activated FAsi. Want to be able to control when you oxidize FA

ii. Carnitine acyltransferase I is inhibited by Malonyl-CoA

e. Oxidation Occurs on the Beta Carbon (C3)i. Dehydrogenase

ii. Enoyl Hydrataseiii. L-3-OH dehydrogenaseiv. B-keto thiolase fatty acyl CoA + acetyl CoA

1. Brings CoA in and bumps 2 carbons off, making acetyl CoA to feed into TCA cycle

v. …repeats until FA is all AcetylCoA: [(# of carbons in FA)/2 – 1]cycles9. Fatty acids are the most efficient means of energy storage

a. ~4x more ATP stored per gram of fat versus glucose storage

b. A 70 kg man could survive ~12 hours on glycogen reserves, ~two weeks on amino acids from muscle protein & ~80 days on fat reserves

10. Oxidation of Unsaturated and Nonsaturated FAs 11. Oxidation of branched chain FA (phytanic acids, from plants)

a. Cant form the double bond if the B carbon has a methyl group Must “move” it to the a-carbon

12. Ketone bodies: soluble energy from fats that can travel freelya. The blood-brain barrier prevents albumin carrying FAs from entering

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b. To deal with this, the liver converts FAs to ketone bodiec. There is no human enzyme to convert Acetyl-CoA to pyruvate. Thus FAs

cannot be used for gluconeogenesis

13. Ketoacidosis a. Ketone bodies build up in the blood

i. Occurs in extreme starvation (due to extreme depletion of OAA, arresting TCA cycle)ii. Also occurs in diabetes due to uncontrolled signal to release unneeded FAs (glucagon

uncontrolled by insulin)

b. Loss of ketone bodies in the urine causes loss of associated Na+ and K+ c. This reduces the buffering capacity of blood, causing a drop in blood pH, or

ketoacidosis14. Respiratory quotient : a measure of how much CO2 is released by a given tissues per

O2 consumeda. Low RQ (0.7 to 0.8): tissue is primarily using FAs and/or proteins for energy

(Palmitic Acid)b. High RQ (1.0): tissues primarily using glucose or ketone bodies

LECTURE 25: FATTY ACID BIOSYNTHESIS AND MODIFICATION

1. The liver can efficiently synthesize fatty acids in the cytoplasm, de novo, from carbohydrate breakdown products

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a. Citrate lyase generates cytoplasmic acetyl-CoA

b. Acetyl CoA carboxylase makes Malonyl-CoA, a committed building block of FAs

i. Active only as a polymer and only when NOT phosphorylated

1. High [citrate] indicates high [acetyl-CoA]

ii. Inactive when as a monomer and when phosphorylated 1. Product inhibition (high Malonyl-CoA)2. High Palmitoyl CoA (which Malonyl CoA is used to make)3. Glucagon

2. Fatty Acid Synthase (FAS)a. FAS uses Malonyl-CoA, Acetyl-CoA and reduced NADPH to form FA chainsb. After meal rich in fat, hormones stimulate FASc. Enzyme exists as a head to tail dimerd. Growing FA chain is held alternatively by -SH of FAS-Cysteine residue or by

FAS-linked phosphopantetheinei. Attach one molecule of

acetyl CoA to FAS-Cysteine residue

ii. Then attach a molecule of malonyl CoA to FAS-linked phosphopantetheine

iii. Transferase activity can move something from the FAS-linked phosphopantetheine to the FAS-Cysteine residue

iv. Heal to tail orientation lines the molecules up for transfer and condensation

e. All condensation adds to the carboxyl end elongating the chain doesn’t change the omega number

f. Synthesis proceeds by two carbon units until 16 carbons (five more times for a total of 7 times)

i. Thioesterase cleaves off C16 palmitate leaving FAS ready for another cycle

g. Sources of NADPH for FA Synthesis i. Pentosephosphate pathway

ii. NADPH-linked malate dehydrogenase (malic enzyme): OAA malate Pyruvate (pyruvate feeds back to mitochondria)

1. Way of regenerating NADPH unique to FA synthesis2. Have effectively converted NADH to NADPH

3. Overall Requirements for FA synthesis (16:0 palmitate)

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a. 8 Acetyl CoAb. 7 ATP (to convert 7 Acetyl CoA to 7 Malonyl CoA)c. 14 NADPH (7 b-keto and 7 enoyl reductions)

4. Carbohydrates are converted efficiently to FAs a. Efficiency = [100%](129 + 9)/(4.5)(38) = 81% Thus, you can easily gain

weight on low fat, high carbohydrate diets5. Modifications of Fatty Acids

a. Elongases : Add 2 carbon units (from Acetyl CoA or Malonyl-CoA) to carboxyl end, followed by reduction & dehydration

b. Desaturases : Add cis double bonds, generally spaced by 3 carbons, in mammals never closer than w-7c. Oxygenases: Oxidize polyunsaturated FAs to create intercellular messengersd. Hydroxylases: Add -OH to alpha carbon (C2) of some FAs used in nervous tissues

6. Fatty Acid Elongation (similar to synthesis but with different enzymes)a. Formation of the b-ketone:

i. On smooth endoplasmic reticulum (SER) 2 carbons from MalonylCoA 1. Palmitoyl-CoA+Malonyl-CoAbketostearoylCoA+CO2+ CoA

ii. In mitochondrial matrix 2 carbons from AcetylCoA 1. Palmitoyl-CoA + Acetyl-CoA b-keto-stearoyl-CoA + CoA

b. Reduction to b-OH using NADPHc. Dehydration to 2,3 enoyld. Reduction to stearoyl-CoA using NADPHe. Elongation adds to carboxyl end- does not change the omega number

7. Fatty Acid Desaturase Mechanism a. Takes molecule from saturated to cis-unsaturated double bonds in SERb. On SER: uses molecular oxygen, NADH and cytochrome b5 to yield a double bond, H2O and NAD+

c. Double bonds generally 3-carbons apart (e.g. 20:5D5,8,11,14,17, EPA)d. Human enzymes can only desaturate at w-7 or greater (further from

terminal methyl)8. Essential Fatty Acids

a. Precursors for all w -3 and w -6 FAs must be gotten in diet:

i. αLinolenic Acid 18:3D9,12,15 w-3ii. Linoleic Acid 18:2D9,12 w-6

b. Sources:i. Linoleic acid - 20-80% of almost all plant oils

ii. αLinolenic acid - Linseed (61%), canola, soybean, walnut (~10%); Other w-3s - Herring oil (~20%)

9. Formation of Triacylglycerol (Neutral Fat)a. Triacylglycerol synthases : glycerol-3-P + 2

acetyl-CoA phosphatidic acid diacylglycerol + acetyl-CoA triacylglycerol

b. Can also turn dietary fat into 2-Monoacylglycerol via pancreatic lipase, and that into triacylglycerol

10. Low carbohydrate, high protein (LCHP) dietsa. Why it might work:

i. Glucagon stimulates FA released from adiposeii. Ketone bodies from FAs increase… mild ketosis may curb

appetiteiii. Protein intake may provide enough AAs for gluconeogensis,

no need for muscle breakdowniv. Without DHAP from glycolysis, TAG synthesis in adipose

is low

11. Hormonal Appetite Controls:

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a. Leptin , released by adipose tissue, suppresses food intake by altering the hormones released from the hypothalamus; downregulates FA synthase

b. In mice, absence of leptin production leads to extreme adiposity (obesity)12. Fat free diets didn’t produce fat free people

a. Obesity: BMI of 30 or higherb. The relative risk of diabetes increases by ~25% for each additional unit of BMI over 22 kg/m2

c. The risk of CHD doubles at BMIs of 25 to 28.9, and triples at BMIs of 29 or greater (relative to BMI of <21)

d. High BMIs correlate withi. Increased cholesterol levels; Decreased HDL levels; Hypertension (high blood pressure)

LECTURE 26. SYNTHESIS AND FUNCTION OF MEMBRANE LIPIDS

1. Lipids are the basis for biological membranes: Hydrophobic core does not allow charged protons to get through; maintains concentration gradient necessary for ETC

2. Glycerophospholipids a. Spontaneously form bilayer vesicles in aqueous solution

3. Cardiolipin: A double phospholipid common on mitochondrial membranes; two phosphatidyl groups linked by a glycerol

4. Nature of attached FAs important for PL function a. Common Lecithin: 1 stearoyl, 2 oleoyl-phosphatidyl choline

i. Most abundant human membrane lipidii. Unsaturated FA on C2 lowers MP fluid membranes

b. Lung Surfactant: 1,2 distearoyl-phosphatidyl choline (2 saturated FAs)i. Reduced fluidity important for coating air-water interface, preventing

alveolar collapse. A deficiency can lead to respiratory distress syndrome in premature infants.

5. Formation of Glycerophospholipids Starts with Phosphatidic acid (PA) or DAG

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a. Use some of the same intermediates used in PG synthesisb. DAG vs. Head Group Activation

i. DAG used for : Inositol (PI), Glycerol@C1(PG), PG@C3 (cardiolipin)ii. Head group used for : Ethanolamine (PE), Choline (PC)

6.

Plasmalogens: Glycerophospholipids with Fatty Alcohol in Ether Linkage at C1 (instead of an ester-linkage)

a. Synthetic pathway differs from that for ester-linked PLsb. Abundant plasmalogens in mitochondrial inner membrane may resist

oxidative damage that would hydrolyze the ester bonds in normal PLsc. E.g. Activates platelet secretion & alters membrane permeability

7. Phospholipases (PLs)a. PLAs in many venoms: lysophospholipids

(missing one FA) act as hemolytic detergents

8. Modified Fatty Acids as Intracellular Signalsa. Certain stresses induce Phospholipase A2s

to release long-chain PUFAs from membrane phospholipids

i. E.g. Arachidonic acid & other long-chain w3 or w6 PUFAs, such as EPA -eicosopentaenoic acid

b. Cyclooxygenase gives rise to prostaglandins & thromboxanes (short-range intercellular signals to promote pain, fever, thrombosis, inflammation)

i. Cyclooxygenase removes 2 double bonds, & that subscript shows # of double bonds remaining (is always 2 less than the original PG)

ii. 20:4 arachidonate gives rise to PG2 (endothelium) & TX2 (platelets) series; 20:5 eicosopentaenoate gives rise to PG3 series

iii. Aspirin & other anti-inflamatory agents block activity of Cyclooxygenase

c. Lipoxygenases generate leukotrienes, important for immune responsesi. # of double bonds is unchanged by 5-lipoxygenase reaction (does not

reduce double bonds)d. DAG and IP 3 released from PIP2 act as intracellular signals/second

messengers

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i. Kinases phosphorylate PI to PIP and PIP2

ii. Extracellular Signal binds membrane-spanning receptor to activate Phospholipase C enzyme which cuts PIP2 into:

1. DAG Fatty acid chains in inner lipid monolayer of plasma membrane and activation of protein kinase A

2. IP 3 activation of Calmodulin Kinase9. Sphingolipids : membrane lipids based on spingosine, not glycerol

a. FA attached through an amide (rather than an ester) forms a ceramideb. Sphingomyelin in myelin sheath with C24 FAs provides electrical insulation

i. Only sphingolipid that is a “phospholipid” (contains phosphate)c. Glycolipids: Glucocerebroside (or Galactocerebroside)

i. Addition of more sugars or sulfate makes Sulfatides, Globosides & Gangliosides

1. e.g. GM2, Tay Sachs ganglioside2. Gangliosides and globosides are markers used in cellular

recognition (such as the blood group antigens)d. Normal Sphingolipid Breakdown Pathways

i. Membrane lipids are constantly pulled from the surface into endosomesii. Some are reused, others are broken down in lysosomes & their components recycled

iii. Each sugar linkage in the glycosphingolipids (cerebrosides, globosides and gangliosides) requires a separate enzyme for removal

e. Lysosomal storage diseases i. Many sphingolipids are most abundant in the brain

ii. Genetic defect in sphingolipid breakdown enzyme leads to slow accumulation of lysosomes full of partial breakdown products

iii. This damages nerve cells and usually leads to progressive neurodegeneration and death youngiv. Screening for Hexosamidase A activity & gene mutations in at-risk parents & fetuses has

reduced frequency of Tay Sach

10. Lipids as membrane anchors for proteins a. Attaching lipid to a protein pulls a cytoplasmically soluble protein to the

membraneb. Allows a cell to decide, under various signaling condition, whether to keep the protein in the cytoplasm,

or attach it to the membrane (versus integral membrane proteins which will always be in the membrane)c. Palmitate (16:0) palmitoylationd. C14 myristate myrystoylatione. C15 isoprenoid farnesine farnesylationf. Modified phoshatidyl inositol GPI anchor

Lecture 27. STEROIDS AND FAT-SOLUBLE VITAMINS

1. Cholesterol a. Important membrane lipid: reduces fluidity/permeability; cholesterol raftsb. Important derivatives: Bile salts, steroids hormones, Vitamin D, coenzyme Qc. Sources:

i. Animal products in diet; Dietary cholesterol goes to liver as CEs in chylomicron remnants

ii. Endogenous synthesis (primarily by liver, but also skin, intestines & kidney)2. Cholesterol Synthesis uses Cytoplasmic Acetyl-CoA & NADPH

a. Similarly to FA Synthesis:i. Acetyl CoA from the Citrate Lyase pathway

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ii. NADPH from Pentose Phosphate pathway & NADPH-linked Malate Dehydrogenase

b. Cytoplasmic Acetyl-CoA is converted to HMG-CoA (Essentially the same as mitochondrial HMG-CoA formation to make ketone bodies)

c. HMG-CoA converted to mevalonic acid i. HMG-CoA reductase is the

major regulated step ii. Inhibited by : (signals that tell

the body to release energy)1. Free cholesterol 2. Phosphorylation

(indirectly controlled by glucagon & epinephrine: cAMP dep. PK activates a second kinase)

3. Statin drugs (which decrease the levels of circulating cholesterol by mimicking substrate/product and bind to the active site of the enzyme)

d. Mevalonic acid (6C) is activated to form IPP (5C)e. Squalene is formed by condensation reactions of isoprene units

i. Head (of DPP- an IPP isomer) to tail (of IPP) condensations, followed by reductive head to head condensation of fanesyl pyrophosphate (15C)

f. Cyclization and maturation of cholesteroli. Squalene Monooxygenase makes a Squalene epoxide

ii. Cyclase holds the epoxide in the right orientation so it can form the four-ringed sterol structure (Lanosterol, 30C)

iii. Demethylation followed by rearrangement forms cholesterol (27C)

3. IPP Condensations Yield Other Important Isoprenoids

a. Ubiquinone uses isoprene units to make its hydrophobic tail units to stick into the membrane

b. Dolichol (a sugar carrier)4. Bile salts (bile acids) are synthesized in

the liver from cholesterol to aid dietary fat absorption

a. Emulsion: hydrophilic shell around the molecule in water allows pancreatic lipase to work

b. 7-α hydroxylase enzyme (7 position is where enzyme starts makes bile salts)

5. Enterohepatic Circulation : Preventing Excessive Cholesterol Lossa. Bile acid & Cholesterol made in the liverb. Released by gall bladder to emulsify dietary fats

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c. Endogenous and exogenous cholesterol taken up and returned to liver as chylomicronsd. Active (energy requiring) uptake of bile salts, for return to liver

6. Control of Circulating Cholesterol and Heart Disease a. When cholesterol is too abundant, levels of circulating LDL increase,

which is a root cause of atherosclerosisb. LDL levels can be controlled by :

i. Ensuring proper LDL uptake by LDL-Receptorsii. Decreasing synthesis (HMG-CoA reductase inhibitors)

iii. Decreasing dietary cholesteroliv. Promoting excretion of excess cholesterol (as bile salts)

7. Steroid Hormones

8. Fat Soluble Vitamins a. Isoprenoids required as cofactors or signaling molecules that cannot be

synthesized de novo or for which de novo synthesis may not be sufficient (e.g.

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Vitamin D)i. A (light reception & hormone)

ii. D (hormone controlling calcium absorption)iii. E (antioxidant)iv. K (important cofactor for blood clotting)

b. "The FAT cat is in the ADEK (attic)": Fat soluble vitamins are A,D,E,Kc. Are hydrophobic enough that they need to be transported in the blood using lipoproteins; Not soluble in

water- cannot be easily excreted

d. Great excess (>10x RDA) can be toxic, especially for D & Ai. Excess of fat soluble vitamins are stored in adipose and can cause health issues

9. Vitamin A a. The three faces: similar structure, differing only at one (terminal) carbonb. Retinal : aldehyde

i. Bound to opsin, forms rhodospsinii. Light induces a cistrans isomerization, causing dissociation and the

conformational change in opsin is the first signal in vision1. Vitamin A deficiency results in night blindness

c. Retinol: alcohold. Retinoic : carboxylic acid

i. Hormone important in growth and differentiation; Activates transcription factors of the steroid receptor class

10. Vitamin D a. Penultimate intermediate in cholesterol biosynthesis has a single double bond at the 7 positionb. Only occurs in the skin; can synthesize with sufficient UV exposurec. Activates Vitamin D hormone receptor transcription factorsd. Promotes intestinal Ca++ absorption and bone formatione. Deficiency and Toxicity both promote demineralization of bones

a. Deficiency : poor calcium absorption, bone demineralization and rickets (in children) or osteomalacia (in adults)

b. Toxicity : excess serum calcium: renal stones & metastatic calcifications; calcium taken from bonesdemineralization

11. Vitamin E a. Important antioxidant; terminates free radical oxidation of unsaturated

FAs by taking the free radical onto itself12. Vitamin K

a. Essential co-factor for blood clotting; allows the γ-carboxy-glutamate to be formed, forming a vitamin K epoxide in the process which can be recycled back to vitamin K hydroquinone

b. Blocking this process results in internal bleedingi. Vit K Epoxide reductase inhibitors (dicourmarins like warfarin &

coumarol) cause uncontrolled bleedingii. Poisons at high doses, lower doses can treat thrombosis (too much

blood clotting)c. Vit. K deficiency can cause hemorrhagic disease in premature infants

13. Integration of Lipid Metabolisma. Hormonal Controls:

i. Glucagon or Epinepherine (blood glucose is low, cells need energy);

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inactivates all the new biosynthesis; Signaling cascade causes protein phosphorylation by cAMP-dependent protein kinase (PKA)

1. Inactivates Acetyl-CoA carboxylase2. Inactivates HMG-CoA reductase (inhibit cholesterol synthesis)3. Inactivates glycogen synthase 4. Activates hormone sensitive lipase (release FA)5. Activates glycogen phosphorylase (release glucose from

glycogen stores)ii. Insulin bound to a receptor tyrosine kinase has multiple opposing

effects (not as simple as removal of phosphates added by PKA)b. Intracellular regulation by small molecules:

i. Cholesterol : 1. Inhibits HMG-CoA reductase ( increases LDL uptake)2. Decreases LDL Receptor level3. Increases ACAT activity (form CE deposits and lipoproteins)

ii. Fatty acyl CoA and Malonyl CoA inhibit Acetyl CoA carboxylaseiii. Citrate activates Acetyl CoA carboxylaseiv. Malonyl CoA inhibits Carnitine acyl transferase I (CATI) (decrease B

oxidation)c. Other limitations:

i. CHOs needed for G3P in adipose to make TAGsii. CHOs needed for OAA to make citrate for FA & cholesterol synthesis

iii. Liver lacks 3-ketoacylCoA transferase (makes but does not use KBs)1. RBCs also cannot use ketone bodies, only can use glucose

iv. Can’t get glucose from FAs (no enzyme to make pyruvate from Acetyl-CoA); Can only use the glycerol backbone from fat to make a small amount of glucose via gluconeogenesis

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