MEE NOTES – Metabolism, energetics and enzymes

Lecture 1 – Introduction to protein structurePROTEIN: any group of organic compounds composed of one or more chains of amino acids and forming and essential part of all living organisms.

• Outline the reaction by which amino acids are joined together.

Condensation reaction.

A water molecule is excluded and 2 amino acids join, carboxyl end to amine end.

Same reaction occurs between polypeptide chains to form longer ones.

• Sketch a trimeric peptide, illustrating the amino -terminus, carboxyl terminus and side chains.

• Appreciate the different types of bond that combine to stabilise a particular protein conformation.

• Distinguish between an α-helix and a β-pleated sheet and appreciate the bonds

that stabilise their formation.

• Understand the concepts of primary structure, secondary structure, tertiary structure & quaternary structure with respect to proteins.

Proteins have structure to fulfil functions which rely on specificity. Functionality requires a defined 3D structure/conformation. Proteins generally possess a degree of flexibility.

PEPTIDE BONDS: no free rotation around bond. C=O and N-H in same place of molecule. Other 2 bonds in backbone of polypeptide are able to rotate.Only conformations where side chains don’t clash with main chain are allowed. Steric hindrance.

Bonds in proteins

Covalent disulphide bridges. Strongest bonds

Hydrogen bonds. Between atoms on different sidechains/backbone or between water molecules.

Ionic interaction (salt bridges). Electrostatic attraction between charged side chains. Relatively strong, particularly when ion pairs are within the protein exterior and excluded from water.

Van der Waals. Transient and weak, due to fluctuating electron cloud producing and inducing dipoles.

Hydrophobic interactions. Major driving force of protein folding. Create a hydrophobic core and a hydrophilic surface in most proteins.

Primary structure: linear sequence of amino acids.

Secondary: local structural motifs within a protein. ALPHA HELICES and BETA PLEATED SHEETS.

Tertiary: the arrangement of the secondary structure motifs into compact globular structures called domains.

Quaternary: the 3D structure of a Multimeric protein composed of several subunits.

• Outline how warfarin works with reference to the post translational modification of glutamate.

WARFARIN

Anticoagulant. Works by inhibiting carboxylation reactions. Clotting cascade require carboxylation (post-translational modification NAGE 5)

Lecture 2 – Enzymes and Energetics

ENZYME: a protein that acts as a catalyst to induce chemical changes in other substances, itself remaining unchanged by the process. Increase speed of a reaction by providing an alternative route for a reaction to take. Reduce activation energy.

• Explain the concept of free energy and how we can use changes in free energy to predict the outcome of a reaction.

FIRST LAW OF THERMODYNAMICS: Energy can neither be created nor destroyed. It’s simply converted from one form to another.

SECOND LAW OF THERMODYNAMICS: In any isolated system (e.g. a single cell/the universe), the degree of disorder (entropy) can only increase.Reaction proceed spontaneously towards products with greater entropy.

FREE ENERGY

THE AMOUNT OF ENERGY WITHIN A MOLECULE THAT COULD PERFORM USEFUL WORK AT A CONSTANT TEMPERATURE. Denoted as G, in kJ/mole

» Combines 1st and 2nd laws.» Change in G (∆ G) measure amount of disorder resulting from a certain reaction.» E.g. ∆G = free energy of products – free energy of reactants» Reaction can happen spontaneously if ∆G is negative. Not if positive.

• Draw the chemical structure of ATP and explain how it acts as a carrier of free energy and is used to couple energetically unfavourable reactions.

Phosphoanhydride bonds have a large negative ∆G of hydrolysis so are HIGH ENERGY bonds.

Coupled Reactions

Pathways within the cell that synthesise molecules are generally energetically unfavourable e.g. peptide synthesis

They take place because they are coupled to an energetically favourable one. Providing that the sum of the ∆G for the overall reaction is still negative, the reaction will

proceed. The majority of energetically unfavourable biochemical reactions rely on the hydrolysis of

high-energy phosphate bonds such as those found in ATP.

E.g. Glucose Fructose

In a biological setting, energetically favourable reactions won’t occur at a useful rate unless catalysed by enzymes.

• Describe how enzymes act as catalysts of reactions with reference to the reactions catalysed by lysozyme and glucose-6-phosphatase.

Enzymes function by lowering the barriers that block a particular reaction.

Enzymes bind one or more substrate molecules tightly within a part of the protein known as the active site.

Enzymes arrange the substrate(s) in such a way that certain bonds are strained. Key residues within the enzyme participate in either the making or breaking of bonds by altering the arrangement of electrons within the substrate(s).

This can often take the form of either oxidation reactions, (in which electrons are removed from an molecule) or reduction reactions (in which electrons are added to a molecule) .

The transition state is the particular conformation of the substrate in which the atoms of the molecule are rearranged both geometrically and electronically so that the reaction can proceed.

Enzymes work by bending their substrates in such a way that the bonds to be broken are stressed and the substrate molecule resembles the transition state.

This makes them more amenable to reaction with other molecules.

LYSOSYME: component of tears and nasal secretions and is one of the first lines of defence against bacteria.

– It catalyses the hydrolysis of sugar molecules within bacterial cell walls that are necessary for their structure. With this bond broken, the bacteria lyse and die. Discovered by SAF.

• Outline the differences between lock and key and induced fit models of substrate-enzyme interactions.

INDUCED FIT: substrate induces a change in the conformation of the enzyme which results in the formation of the active site. Enzyme reverts back to original conformation when products released.

This is the correct model proteins generally possess a degree of flexibility necessary for function.

LOCK AND KEY: shape of substrate matches the active site of the enzyme. Explains the specificity of most enzymes for a single substrate.

• Describe graphically, the effects of substrate concentration, temperature and pH on reactions catalysed by enzymes.

TEMPERATURE

» Chemical reactions speed up as temperature is increased, so, in general, catalysis increases at higher temperatures.

» However, each enzyme has a temperature optimum, beyond which its conformation is said to be denatured and the enzyme is inactive.

pH

» Most enzymes have an optimum pH for their activity. » The catalytic side chains are in the correct state of ionisation.

Lysozyme, optimum pH 5.0

• Illustrate the role of the coenzyme NAD in the reaction catalysed by lactate dehydrogenase.

NAD+ (Nicotinamide adenine dinucleotide) is a vital component of many dehydrogenation reaction within the body.

No effect on its own, functions only by binding to a protein. COFACTOR IN DEHYDROGENATION REACTIONS. Like enzymes, they differ with respect to their substrate specificity.

Catalyses dehydrogenation by readily accepting a hydrogen atom and 2 electrons.

» During intense exercise, skeletal muscles function anaerobically, oxygen is a limiting factor. Metabolite pyruvate is converted into lactate. This generates free NAD+ which is needed by the muscle for other reactions.

» Lactate diffuses from the muscle into the blood stream and is picked up by the liver, where the high levels of NAD+ can be used by lactate dehydrogenase to regenerate pyruvate.

Lecture 3 – Metabolic pathways and ATP production-1

• Sketch a cartoon of the three stages of cellular metabolism that convert food to waste products in higher organisms, illustrating the cellular location of each stage.

Digestion

Enzyme mediated, liberates small molecules, takes place in the intestines.

Cellular Metabolism I

Oxidation of the small molecules within the cytosol of individual cells, generating ATP and NADH.

Cellular Metabolism II

Oxidation of the small molecules generated by the first stage of cellular metabolism within the mitochondria of individual cells, generating ATP and waste products.

• Outline the metabolism of glucose by the process of glycolysis, listing the key reactions, in particular those reactions that consume ATP and those that generate ATP.

GLYCOLYSIS: An anaerobic process occurring the cytoplasm of the cell. 1 6 carbon molecule (glucose) 2 3 carbon molecules (pyruvate)

•

REACTIONS:

All kinases catalyse the transfer of a phosphate group from a donor such as ATP onto a substrate.

Rn. 2 Isomerisation shuffles glucose chair to give fructose. Fructose can be split into 2 equal halves.

Rn. 3 Fructose-1,6-bisphosphate is highly symmetrical and high energy. Regulation of phosphofructokinase controls sugar entry into glycolysis pathway.

Rn. 4 opening fructose ring generates 2 high energy compounds.

Rn. 5 deficiency in triose phosphate isomerase extremely rare, most suffers die within 6 years.

Rn. 6 NADH generates used to generate more ATP in oxidative phosphorylation.

Rn. 7 Phosphate group transferred to ADP to give ATP.

Rn. 8 Mutase phosphate group shuffled from 3 position to 2

Substrate level phosphorylation: the production of ATP by direct transfer of high-energy phosphate group from intermediate substrate in biochemical pathway to ADP. E.g. glycolysis.

Differs from oxidative phosphorylation where ATP is made using energy from electron transfer in ETC.

• Distinguish between the aerobic and anaerobic metabolism of glucose with reference to the enzymes involved and the comparative efficiencies of each pathway with respect to ATP generation.

» From anaerobic metabolism of one molecule of glucose we only generate 2 molecules of pyruvate and 2ATP molecules (net).

» This contrasts poorly to the complete aerobic metabolism of a molecule of glucose which can theoretically yield 38 molecules of ATP.

ANAEROBIC CONDITIONS – characteristic of yeasts.

Alcoholic Fermentation

Pyruvate Acetaldehyde Ethanol

H+ CO2 NADH + H+ NAD+

Enzyme – pyruvate decarboxylase Enzyme – alcohol dehydrogenase

Lactate Generation

Pyruvate Lactate

NADH + H+ NAD+

Enzyme – lactate dehydrogenase

Both alcoholic fermentation and lactate generation allow NAD+ to be regenerated, and so let glycolysis continue. E.g. needed for the dehydrogenation of glyceraldehyde 3-phosphate. First step in ATP generation.

Pyruvate + HS-CoA Acetyl CoA + CO 2

NAD+ NADH

Enzyme: Pyruvate dehydrogenase complex

Series of reaction occur in the MITOCHONDRIA. Acetyl CoA formed in committed to entry to the TCA cycle to ultimately produce ATP in oxidative phosphorylation.

• Describe the reactions catalysed by lactate dehydrogenase and creatine kinase and explain the diagnostic relevance of their appearance in plasma.

LACTATE DEHYDROGENASE present in many tissues, heart, liver, kidney…

Elevated levels can be diagnostic of:

Stroke Heart attack Liver disease (hepatitis) Muscle injury Muscular dystrophy Pulmonary infarction

Creatine Phosphate

During exercise, amount of ATP in muscle can only sustain contraction for around 1 second.

Large reservoirs of creatine phosphate buffer the demand for phosphate.

Creatine Phosphate Creatine + ATP

ADP + H+ ATP

Enzyme – creatine kinase

Creatine kinase leaks into bloodstream upon muscle damage.

Elevated levels can be used to:

Diagnose myocardial infarction Determine extent of muscle damage Evaluate cause of chest pain Discover carriers of muscular dystrophy.

• Outline the oxidative decarboxylation reaction catalysed by pyruvate dehydrogenase, with reference to the product and the five co-enzymes required by this enzyme complex.

PYRUVATE DEHYDROGENASE

Gigantic, consists of 3 individual enzymes and 5 co-factors. THIAMINE PYROPHOSPHATE (TPP), LIPOAMIDE, FAD, CoA and NAD+ (cofactors)

Enzymes with prosthetic groups: pyruvate decarboxylase with TPP, lipoamide reductase with lipoamide, dihydrolipoyl dehydrogenase with FAD

Prosthetic groups such as lipoamide are a permanent part of the complex, whereas NAD+ and other co-factors bind reversibly to enzymes.

Lecture 5 – Metabolic pathways and ATP production – II

• Describe the processes by which the fatty acid palmitate and the amino acid alanine are converted into acetyl-CoA.

ALANINE – undergoes transamination by action of alanine aminotransferase

Pyruvate TCA cycle. Glutamate reconverted to alpha-ketoglutarate.

• Outline the Krebs or TCA (tricarboxylic acid cycle) with particular reference to the steps involved in the oxidation of acetyl Co-A and the formation of NADH and FADH2 and the cellular location of these reactions.

KREBS: aerobic as NAD+ and FAD only regenerated by electron from O2 in ox. phosph.

Oxidation of 1x Acetyl CoA 3 x NADH 1x FADH2 1x GTP = 12ATP 38 ATP per glucose molecule

TCA enzyme location: Soluble proteins located in MITOCHONDRIAL MATRIX SPACE

Except for succinate dehydrogenase which is an integral membrane protein attached to inner surface of inner mitochondrial membrane. Can communicate directly with components in repiratory chain.

» Majority of energy from food metabolism is from the re-oxidisation of reduced coenzymes in respiratory chain. Inner mitochondrial membrane…oxidative phosphorylation.

NADPH takes part in anabolic reactions, whereas NADH takes place in catabolic reactions.

The use of different co-factors for sets of reactions allows electron transport in catabolism to be kept separate to that of anabolism.

• Outline the glycerol phosphate shuttle and the malate-aspartate shuttle, in particular stating why these mechanisms are required.

Glycerate phosphate shuttle

Electrons from NADH, rather than NADH itself are carries across the mitochondrial membrane via shuttle.

1. Cytosolic glycerol 3-phosphate dehydrogenase transfers electrons from NADH to glycerol 3-phosphate.

2. A membrane bound form of the same enzyme transfers the electrons to FAD. These then get passed to co-enzyme Q, part of the electron transport chain (next lecture).

Malate-Aspartate Shuttle

System uses 2 membrane carriers and four enzymes.

Net reaction: NADH cytoplasmic + NAD+ mitochondrial NAD+ cytoplasmic + NADH mitochondrial

H- transferred from cytoplasmic NADH to oxaloacetate to give malate, catalysed by malate dehydrogenase (MDH)

Malate can be transported into mitochondria where it’s re-oxidised by NAD+ giving oxaloacetate and NADH. Catalysed by mitochondrial MDH.

A = Alpha-ketoglutarate tranporter. Exchanges alpha-ketoglutarate for malate.

B = glutamate/aspartate transporter.

Exchanges glutamate for aspartate.

• Calculate the theoretical maximum yield of ATP per glucose molecule oxidized by aerobic respiration and compare this to the theoretical maximum yield of ATP per molecule of palmitic acid.

• Give two examples of the use of NADPH in reductive biosynthesis

1. Biosynthesis of RNA2. Biosynthesis of cholesterol. Reduced C=C by transfer of hydride ion

NADP+ is an electron carrier. Can pick up 2 high energy electrons and a proton. Collectively known as a hydride ion (H-).

NADP+ doesn’t take part in electron transfer but gives a slightly different conformation so it will bind to different enzymes than NAD+

Lecture 5 – Mitochondria and oxidative phosphorylation• Draw a cross sectional representation of a mitochondrion, and label its component parts.

Oxidative phosphorylation takes place on the inner membrane, unlike Krebs which takes place in the matrix.

Numerous cristae folds increase the SA where oxidative phosphorylation can take place.

• Outline the proposed evolutionary origins of mitochondria.

Mitochondria:

» Believe to be evolutionary descendants of a prokaryote. Endosymbiotic relationship established with eukaryotic cell ancestors.

» Thought to have occurred early in history of life. » Many genes needed for mitochondrial function translocated to nuclear genome.» Genome of Rickettsia prowazekii revealed several genes are closely related to mitochondrial

genes.

• Outline the chemiosmotic theory.

Chemiosmotic Hypothesis of Oxidative Phosphorylation

Oxidative phosphorylation proceeds in two steps:

1) The translocation or movement of protons from within the matrix of the mitochondria. This is controlled by the electron transport or respiratory chain.

2) The pumped protons are allowed back into the mitochondria through a specific channel, which is coupled to an enzyme which can synthesise ATP known as ATP synthase.

The proton motive force that drives H+ back into the matrix space consists of a pH gradient and a transmembrane electrical potential

• Describe the electron transport chain in mitochondria with reference to the functions of coenzyme Q (ubiquinone) and cytochrome c.

MEMBRANE COMPLEXES:

NADH Dehydrogenase complex Sytochrome b-c1

complex Cytochrome oxidase

complex

MOBILE CARRIERS

Ubiquinone (co-enzyme Q)

Cytochrome C

These proteins accept electrons and in doing to, a H+ from aqueous solution.

UBIQUINONE

» Can pick up 1 or 2 electrons with a H+ from solution and passes them to cytochrome b-c1 complex

» Hydrophobic tail confines it to lipid bilayer where it’s needed.

CYTOCHROME OXIDASE

» Receives 4 electrons in final electron transfer step from cytochrome c. Passes electron to oxygen to make water.

» 4 H+ also pumped into intermembrane space, enhances proton gradient.

STEPS

1. Electrons from NADH transferred to ubiquinone by NADH dehydrogenase complex. H+ transferred across membrane to intermembrane space.

2. Ubiquinone carrier electrons to cytochrome b-c1 complex. More H+ carried from inside to outside the membrane.

3. Electrons also transferred from FADH2 to ubiquinone with H+ transferred across the membrane.

4. Cytochrome C transfers electrons to cytochrome Coxidase complex.5. Cytochrome oxidase complex transfers electrons from cytochrome C to oxygen, the terminal

electron acceptor. Water formed as product.

WATER is the perfect terminal electron acceptor as it’s got a high electron affinity. Provides driving force for oxidative phosphorylation.

Proton motive force generated across inner membrane of mitochondria.

• Describe how ATP synthase is able to generate and utilise ATP respectively, with reference to its structure.

ATP SYNTHASE

As membrane are impermeable to ions, any H+ going back into the mitochondrial matrix goes via channel ATP synthase. Energy derived from the movement of the proton goes into phosphorylating ADP to produce ATP. Oxidative phosphorylation

Multimeric enzyme consenting of F0

membrane bound part and F1 part that protrudes into matrix space.

As hydrogen ions flow through pore in membrane, rotation drives transitions of the catalytic portions of the beta subunits, altering affinities for ATP and ADP.

Torsional energy flow from the catalytic subunit into ADP and Pi bound to the channel.

Promotes ATP formation.

Explain why carbon monoxide, cyanide, malonate and oliogomycin are poisonous in terms of their effects on specific components of the electron transport chain.

Metabolic Poisons

CARBON MONOXIDE

Binds with Fe2+ form of haem group and blocks the flow of electron. Behaves like Cyandine

CYANIDE: SUPERTOXIC.

Binds with a high affinity to Fe3+ in the haem group of cytochrome oxidase complex. Blocks flow of electrons through respiratory chain and thus ATP production.

MALONATE: acts as a competitive inhibitor of succinate dehydrogenase (Krebs enzyme in the inner mitochondrial membrane. Passes electrons directly to ubiquinone via FAD.

Slows the flow of electrons from succinate to ubiquinone by inhibiting the oxidation of succinate to fumarate.

OLIGOMYCIN: antibody produced by Streptomyces. Inhibits oxidative phosphorylation by binding within ATP synthase stalk.

Blocks flow of protons through ATP synthase. ATP synthesis inhibited, backlog of protons in intermembrane space. Eventually, this inhibits the flow of electrons through ETC as H+ outside mitochondrion will

build up to saturation point. No more protons can be pumped out against proton gradient.

DINITROPHENOL: induces weight loss by transporting protons across mitochondrial membrane, uncouples oxidative phosphorylation from ATP production. Increases metabolic rate and body temperature.

Margin between slimming does and lethal dose is very slight.

Non-shivering thermogenesis

Regulated uncoupling of oxidative phosphorylation in newborn humans and hibernating animals. UCP – 1/ thermogenin activated in response to drop in core body temperature.

Like DNP, allows protons to bypass ATP synthase, thereby releasing heat from dissipation of proton gradient.

Lecture 6 – Lipid Metabolism

• Appreciate the chemical composition of unsaturated and saturated fatty acids.

Fatty Acids: composed of both hydrophobic and hydrophilic components

They can either be saturated or unsaturated

» Stored in the cytoplasm in the form of triacylglycerol compounds» Specialised cells called adipocytes in mammals take on role of fatty acid storage.» Fats are derived from the diet, de novo synthesis in the liver and storage depots in

adipocytes

BILE SALTS are generated by the liver and stored in the gall bladder.

• Pass from bile duct to intestines during digestion.• Emulsify fats and aid the digestion and absorption of fats and fat soluble vitamins (A,D,E and

K)• Lack of bile salts leads to fatty stools as fat passes through gut undigested.

• Describe the reactions by which the fatty acid palmitate is metabolised to give acetyl-CoA.

β OXIDATION

• occurs in the mitochondria• many stages, ultimately resulting in the production of Acetyl CoA

FATTY ACID + ATP + HS-CoA ACYL CoA + AMP + PP i

Enzyme: Acyl CoA synthetase

Acyl CoA generated undergoes a sequence of oxidation, hydration, oxidation and thiolysis reactions (collectively called beta oxidation)

PRODUCES: 1 X ACYL COA + 1 X ACYL COA SPECIES that’s 2 carbons shorter than the original.

CARNITHINE SHUTTLE:

• generation of Acyl CoA occurs on the outer mitochondrial membrane.

• Acyl CoA needs transporting into the matrix, coupled to carnitine to form acyl carnitine.

• Carnitine and Acyl carnitine are moved to and from the matrix by translocase enzyme

During one cycle, one FADH2 and one NADH are produced. Overall β oxidation of palmitoyl CoA:

Palmitoyl CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA 8 Acetyl CoA + 7 FADH2 + 7 NADH

Glucose metabolism produces 38 ATP

Palmitate metabolism produces 129 ATP.

• Give an overview of the reactions by which fatty acids are synthesized from acetyl-CoA.

Fatty acid metabolism leads to Acetyl CoA production.

• Compare and contrast the pathways for synthesis and metabolism of fatty acids with respect to the substrates and products, coenzymes used, carrier molecules and their cellular location.

FATTY ACID BIOSYNTHESIS = Lipogenesis

» Acetyl CoA Carboxylase» Fatty acid synthase (FAS) – polypeptide with seven different enzymatic activities.

Step#1 The formation of the 3C species Malonyl CoA catalyzed by Acetyl Co-A carboxylase.

Step#2 The transfer of Malonyl from Malonyl CoA-to acyl carrier protein (ACP) to form Malonyl-ACP (catalyzed by Malonyl-CoA-ACP transferase).

Step#3 The transfer of acetyl from a CoA species to acyl carrier protein (ACP) to form Acetyl-ACP (catalyzed by Acetyl-CoA-ACP transferase).

Acetyl-ACP is the initial two carbon unit with which the initial malonyl-ACP formed in step #1 is condensed to form the 4C-species acetoacyl-ACP (a.k.a. β-ketoacyl-ACP).

Step #4 Condensation of acetyl ACP with malonyl-ACP to form a 4C fatty acid species (catalysed by β-ketoacyl ACP synthase). CO2 is generated.

Step #5 Reduction of Acetoacetyl-ACP to D-3-hydroxylacyl-ACP (catalysed by β-ketoacyl ACP reductase).

Step #6 Dehydration to crotonyl ACP (a.k.a. trans-∆2-Enoyl-ACP which is catalysed by 3 hydroxyacyl-ACP dehydrase).

Step #7 Enoyl-ACP reductase catalyzes further reduction to butyryl-ACP.

The process cycles a further seven times from step 4-7 to yield the 16C species palmitoyl-ACP, which is hydrolyzed to give palmitate and ACP.

Overall reaction

Acetyl CoA (C2) + 7 Malonyl CoA (C3) + 14 NADPH +14 H+ Palmitate (C16) + 7 CO2+ 6 H2O + 8 CoA-SH + 14 NADP+

Elongation of the acyl group to make fatty acids longer than 16 carbons occurs seperately from palmitate synthesis in the mitochondria and endoplasmic reticulum (ER).

Desaturation of fatty acids requires the action of fatty acyl-CoA desaturases

The enzyme that creates oleic acid and palmitoleic acid from stearate and palmitate, respectively, is called a ∆-9 desaturase, as it generates a double bond nine carbons from the terminal carboxyl group.

• Give two examples of inborn errors of lipid metabolism with reference to the molecular defects underlying pathology.

Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD)

» Autosomal recessive. Predominantly occurring in Caucasians.» Occurs 1 in 10,000 live births in the UK per year.» If undiagnosed, can be fatal. Thought to account for 1 in100 deaths from Sudden Infant

Death Syndrome (SIDS). » If diagnosed, patients should never go without food for longer than 10–12 hours (a typical

overnight fast). Adhere to a high carbohydrate diet.» Patients with an illness resulting in appetite loss or severe vomiting may need i.v. glucose to

make sure that the body is not dependent on fatty acids for energy.

Primary Carnitine deficiency

» Autosomal recessive disorder. » Occurs 1 in 100,000 live births in the USA per year (1 in 40,000 live births in Japan; 1 in 500

in the Faroe Islands).» Symptoms appear during infancy or early childhood and include encephalopathies,

(cardiomyopathies, muscle weakness; and hypoglycaemia). » Mutations in a gene known as SLC22A5 which encodes a carnitine transporter result in

reduced ability of cells to take up carnitine, needed for the β-oxidation of fatty acids.

Lecture 7 – Cholesterol

•Outline the synthesis of cholesterol from acetyl CoA.

CHOLESTEROL:

» A steroid.» Increases/decreases membrane stiffness, depending on temperature.» Changes interactions with cytoskeleton

Cholesterol, is synthesized via a pathway which can be split in three main parts:

1. Synthesis of mevalonate, a reduced C6 species from 3 Acetyl-CoA units.

2. Activation of mevalonate to isopentenyl-PP (isoprene unit), a C5 precursor which is elongated to squalene, a C30 intermediate species.

3. Cyclisation and demethylation of squalene by monooxygenases to give cholsterol.

All physiological requirements for cholesterol biosynthesis are supplied by the liver. De novo synthesis of cholesterol from acetyl-CoA

Reaction 1-2: 3x Acetly-CoA molecules are combined to generate HMG-CoA

Reaction 3: HMG-CoA reduced by HMG-CoA reductase to generate mevalonate.

Mevalonate undergoes sequential phosphorylation at hydroxyl groups at positions 2 and 5. Forms Mevalonate 2-phospho-5-pyrophosphate.

Mevalonate 3-phospho-5-pyrophosphate decarboxylated forming isopentenyl pyrophosphate. ISOMERISATION REACTION. 3,3-dimethyl-PP produced which can condense with one unit of isopentenyl-PP forming geranyl-PP

3rd Isopentenyl-PP added to from Farensyl-PP. 2 condense to form squalene and 2 molecules of pyrophosphate.

Reaction driven by reducing power of NADPH.

SQUALENE: cyclized to cholesterol.

#1 Squalene reduced in presence of oxygen and NADPH forming squalene 2,3 epoxide.

#2 Squalene epoxide lanosterol-cyclase enzyme catalyses formation of Lanosterol. Series of 1,2-methyl group and hydride shifts forom 4 rings.

#3 Lanosterol reduced and 2 methyl units removed. Generates cholesterol.

Precursor prefnenolone generated from cholesterol by action of desmolase enzyme. 5 classes of steroid hormone are derived from pregnenolone.

VITAMIN D can be made from cholesterol. Exposure to sunlight needed to initiate reaction scheme. Calcitrol plays a key role in Calcium metabolism for vitamin D synthese. Calcium needed to absorb vitamin D.

•Outline the synthesis of bile acids and steroid hormones from cholesterol.

BILE SALTS: major break-down product of cholesterol.

» Generated by the liver» Stored in gall bladder» Pass from bile duct to intestine during digestion» Emulsify fats, aiding digestion and fat absorption.» Lack of bile salts results in fatty stools. (Steatorrhea)

Converted via a series of reactions. Primary bile salts glycocholate and taurocholate.

•Describe the mechanism of transport of cholesterol around the body and its uptake into cells.

LIPID RAFTS: fluctuating assemblies of cholesterol and sphingolipids. Organise cellular signalling by localising key proteins such as cell surface receptors.

Attachment of cholesterol to HEDGEHOD SIGNALLING PROTEIN (N-Hh) limits diffusion in tissues. Key to successful limb formation during embryogenesis.

LIPOPROTEINS:

» Phospholipid monolayer with cholesterol and apoproteins.

» In core of lipoproteins are cholesterol esters and triacylglycerol.

» Insolubility of lipids in aqueous solution poses transportation problems for the body. Packaged into lipoproteins.

» Cholesterol esters more hydrophobic than cholesterol so pack more tightly in lipoprotein core.

Lipoprotein lipase

– Chlyomicrons (DMS) travel from lacteals of intestine to thoracic duct and to left subclavian vein where they enter the bloodstream.

– Enzyme is located on capillary endothelial cells. Links variety of tissues, like adipose, heart and skeletal muscle.

– Catalyses hydrolysis of triacylglycerols in chylomicrons to glycerol and fatty acids.– Fatty acids undergo beta oxidation. Glycerol returned to liver for gluconeogenesis.

•Draw a diagram of low density lipoprotein (LDL) particle and its receptor (LDLR).

LDLs – ‘bad cholesterol’. Prolonged elevation in LDL levels leads to atherosclerosis. Transport cholesterol from liver to peripheral tissues. More than 40% weight made up of cholesterol esters.

LDL Receptor

Receptor mediated endocytosis of LDL

•Explain how mutations of the LDLR give rise to familial hypercholesterolaemia (FH)

INHERITED, MONOGENIC DOMINANT

Heterozygotes have cholesterol levels 2-3 times higher than normal. Susceptibleto atheroscelerosis.

Homozygotes serum cholesterol levels 5 times higher than normal. Sever atherosclerosis and coronary infarction may be observed in adolescence.

Caused by mutation in LDL receptor gene.

CLASS LOCATION OF MUTATION RESULTSI LDLR

promoter/frameshift/deletionLDLR not synthesised

II Throughout coding region LDLR not properly transported from ER to Golgi. Low cell surface expression.

III Mutation in region encoding N-terminus

LDLR doesn’t bind to LDL effectively.

IV Mutation in cytoplasmic domain

LDLR:LDL complex doesn’t cluster in clathrin-coated pits for receptor mediated endocytosis.

V Mutation in EGFP domain LDL not release from receptor in endosome and LDLR nor recycled back to cell surface.

• Give examples of pharmacological agents that may be used to control cholesterol metabolism.

Controlling Hypercholesterolaemia

HMG-CoA-Reductase Inhibitors

E.g. Statins. Lovastatin a competitive inhibitor,

Resins or Sequestrants

Cholestyramine. Bind or sequester (isolate/hide away) bile acid-cholesterol complexes, preventing their reabsorption into the blood stream.

Lecture 8 – Membrane Trafficking

Typed of intracellular transport

1. Gated transport (nuclear import)2. Trans-membrane transport (e.g. import of newly synthesized protein in to the ER)3. Vesicular transport (e.g. inter-organellar transport)

• Explain the terms “endocytosis” and “exocytosis”.

Endocytosis: process by which a cell absorbs molecules by engulfing them.

Exocytosis: process by which a cell directs the content of secretory vesicles out of the cell membrane and into extracellular space.

• Describe the pathway and cellular locations for synthesis, post-translational modification and exocytosis of a secreted protein.

Translocation of newly synthesized proteins into the ER

Common pool of ribosome used to make both proteins staying in cytosol and those transported to ER.

ER signal peptide on newly formed protein directs the engaged ribosome to ER membrane.

mRNA molecules may stay permanently bound to ET as part of polyribosome. Ribosomes moving along mRNA are recycled. At the end of each round of protein synthesis, ribosomal sub units are released and re-join the common pool in the cytosol.

ER: Post translational modifications and quality control

» Folding» Disulphide bond formation» Glycosylation» Proteolytic cleavage» Assembly of Multimeric proteins

Unassembled or misfolded proteins are retained in the ER and exported back into cytosol and degraded.

Sorting at the Trans Golgi Network (TGN): EXOCYTOSIS

• Distinguish “constitutive” and “regulated” secretion.

Constitutive: proteins are continuously secreted from the cell regardless of environmental factors. No external signals needed to initiate the process.

Regulated: proteins are packaged and only secreted in response to a specific signal, such as hormonal or neural stimulation. Cells using this type of secretion normally polarised, secreting multiple classes of proteins. E.g. goblet cells, beta cells on pancreas…

• Describe the process of receptor-mediated endocytosis and the roles played by endocytic vesicles, early endosomes, late endosomes, and lysosomes.

Diagram in previous lecture

• Give a general description of the molecular mechanisms of vesicular transport within cells.

Vesicular transport complexity

Endoplasmic reticulum Golgi apparatus Secretory vesicle/lysosome/endosome

1. Cargo sorting and vesicle formation

2. Vesicle movement3. Vesicle tethering/docking4. Vesicle fusion

• Give examples of diseases resulting from defects in the secretory and endocytic pathways

CFTR – cystic fibrosis transmembrane conductance regulator

• Mutations of the CFTR gene affect functioning of the chloride channels in these cell membranes, leading to CF

• The most common mutation (ΔF508) results from deletion (Δ) of three nucleotides which causes loss of the phenylalanine at the 508th position on the protein

• As a result, the CFTR does not fold normally and is degraded in the ER. No chloride ion channels causes thick, sticky mucus.

Robinow syndrome – dysmorphic facial features, dwarifism, congential heart defects, genital hypoplasia.

Cell surface tyrosine kinase receptor (ROR2). Needed for aspects of cartilage and bone growth. » Mutation in ROR2 causes rethention and degradation in ER.

CONCLUSIONS:

1. Membrane trafficking is a critical process for any cell. Each cell shows many trafficking steps. Main ways are the secretory and endocytic pathways.

2. Molecular mechanisms underlying trafficking are very complex. Vesicular transport is the main mechanism, involved many different proteins.

3. Intracellular trafficking is involved in many diseases; genetic, infection, cancer…

Lecture 9 – Integration of Metabolism

METABOLISM = the sum of all processed in the body. Measurable as oxygen uptake, carbon dioxide release and heat production.

• Outline general features of metabolic activity in liver, brain, muscle, adipose tissue and endocrine pancreas

General metabolic features of some specialised tissues:

• Muscle (40 % of total body weight) can have periods of very high ATP requirement during vigorous contraction and rely on carbohydrate and fat oxidation.

» Capable of large and rapid increases in ATP demand during exercise.» In light contraction, demand met by oxidative phosph. » Anaerobic condition, pyruvate lactate.

• Brain and nervous tissue (2 % of total body weight) uses 20 % of resting metabolic rate as it has a continuous high ATP requirement; cannot utilise fats.

» Glucose dependant metabolism. Needs a continuous glucose supply.» Hypoglycaemia faintness and coma» Hyperglycaemia can cause irreversible damage.» Ketone bodies can partially substitute for glucose

• Adipose tissue (15 % of total body weight) is long term storage site for fats.

• Heart (1 % of total body weight) 10 % of resting metabolic rate and can oxidise fats and carbohydrate.

» Designed for completely aerobic metabolism, large number of mitochondria» Uses TCA cycle substrates like fatty acids and ketone bodies» Loss of oxygen can cause myocardial infarction. Energy demand > supply.

• Liver (2.5 % of total body weight) 20 % of resting metabolic rate; the body’s main carbohydrate (glycogen) store and source of blood glucose.

» Immediate recipient of nutrients absorbed in intestine.» Many metabolic processes (glycolysis, gluconeogenesis, glycogen synthesis)» Highly metabolically active

» Maintains bloody glucose» Lipoprotein metabolism

CONTROL OF METABOLISM

Gluconeogensis

– Making glucose or glycogen from oxaloacetate (Krebs cycle intermediate) – Only a function of the liver– Needs ATP hydrolysis– Not exact reversal of glycolysis, different enzymes bypass some irreversible glycolysis steps.

• Know the effects of eating and fasting on metabolism

EATING

» Increased insulin secretion (causes glucose uptake from blood). Reduced glucagon.» Increased glucose uptake by liver, nor glycogen synthesis and glycolysis» Increased glucose uptake and glycogen synthesis in muscles» Increased triglyceride synthesis in adipose tissue» Increased usage of metabolic intermediates.

AFTER EATING

» Increased glucagon secretion (converts glycogen to glucose). Reduced insulin.» Glucose production in liver, from glycogen breakdown and gluconeogenesis» Use of fatty acid breakdown as alternative substrate for ATP production.

FASTING

» Glucagon/insulin ratio increase further (more glucagon)» Adipose tissues begin to hydrolyse triglycerides to provide fatty acids for metabolism» Krebs cycle intermediates reduced in amount, provides substrate for gluconeogenesis.» Protein breakdown provides amino acid substrates for gluconeogenesis.» Ketone bodies produced from fatty acids in liver. Partially substitute brains need for glucose.

• Describe glucose interactions with lipid and amino acid synthesis & breakdown

• Know basic details of metabolism in muscle

• Know basic details of diabetes as e.g. of dysregulation of metabolism

Type I – inability to make insulin

Type II – reduced responsiveness to insulin and impaired islet function

Metabolism is controlled as if for starvation, regardless of dietary intake. Complications:

» Hyperglycaemia progressive tissue damage» Increase in plasma fatty acids and lipoproteins with possible CV complications» Increased ketone bodies, acidosis» Hypoglycaemia, coma if insulin dosage not controlled properly.