In Class Quiz 3

Define: Lipids, What are their roles?

Define: Lipids, What are their roles?

Water-insoluble molecules that are soluble in organic solvents

Roles Include:

• Membranes

• Energy Storage

• Signalling

• Fat-soluble vitamins

Define: Fatty Acids

Hydrocarbon chains ending with carboxylic acid groups

Key Roles:

• Used for fuel

• Act as building blocks for membrane lipids

How do saturated and unsaturated fatty acids differ?

Saturated: Only single bonds

Unsaturated: One or More Double Bonds (Double bonds may be CIS or TRANS)

What are some ways to name fatty acids?

Systematic: all-cis-Δ9 , Δ12 , Δ15-octadecatrienoate

Common: alpha-Linolenate

Number of Carbons/Number of Unsaturated Bonds: 18:3

Is the conformation of double bonds usually cis or trans?

Cis

What determines the properties of fatty acid chains and lipids?

Chain length and degree of saturation

How does chain length affect melting point?

Longer chains have higher melting points

How does saturation affect melting point?

Unsaturated (double bonds) decrease melting points (when compared to saturated acids with the same chain length)

How are fatty acids stored?

Triacyclglycerols

Descibe: Triacylglycerols

• Efficient storage - hydrophobic and nearly anhydrous

Adipose cells are specialized for storage and mobilization of triacylglycerols. Adipose tissue also provides insulation

What are the components of glycerolipids?

• Fatty acid(s)

• Platform (glycerol, sphingosine, cerebroside)

• Phosphate (usually)

• Alcohol (usually)

What are the components of sphingolipids?

• Fatty acid

• Sphingosine

• Phosphate

• Alcohol

What are the components of glycolipids?

• Fatty acid

• Cerebroside (glycolipid)

• Sugar unit (glucose or galactose)

What are the components of phosphoglycerolipids?

• Fatty acids

• Glycerol

• Phosphate

• Could have alcohol, or no alcohol

Describe: Steroids

• Most common steroid: cholesterol

• Important for membrane fluidity

• Not found in prokaryotes, but in all animal membranes

Define: Liposomes

Lipid vesicles, made from a bilayer membrane that surrounds an inner aqueous compartment

Describe: Eukaryotic Membranes

Membranes contain lipids and proteins, carbohydrates can be attached to lipids or proteins.

Singer and Nicholson proposed the Fluid-Mosaic Model of membranes

What factors affect membrane fluidity?

Heat increases fluidity

Cold decreases fluidity

Unsaturated membranes are more fluid

Saturated membranes are more rigid

Describe: The permeability of lipid bilayers

Lipid bilayers are impermeable to ions and most polar molecules.

This allows the concentrations of an ion inside and outside the cell to be very different e.g. for sodium, 14 mM and 143 mM.

The control of transport across membranes, is one of the key functions of the membrane (barrier). Ion gradients are important for cells.

Describe: Lipid Modifications of Proteins

These modify the biochemical properties of the protein.

• Generally, lipid modifications allow for the association with a hydrophobic environment like the membrane.

• The lipid portion can insert into the hydrophobic interior of the membrane.

• The protein is then localized to the membrane surface for its function

• Immediate donor of free energy, but not long term storage

Describe: Membrane Proteins

Plasma membranes need to conduct the traffic of molecules in/out of cells, they contain proteins that do this.

Membrane proteins can be integral, peripheral, or can be covalently attached via a lipid molecule.

What do most membrane proteins use to cross the membrane?

α helices, they are non-polar and associate with the hydrocarbon core of the lipid bilayer

Describe the use of β strands to cross the membrane

The β strands form a single β sheet with a pore in the center.

ex) porin from the outer membrane of bacteria

True or False: Just part of the protein can be embedded in the membrane, they do not always cross the membrane

True. prostaglandin H2 synthase-1 (outer membrane) is an example of this.

Describe: prostaglandin H2 synthase-1

• Catalyzes the conversion of arachidonic acid into prostaglandin H2

• Arachidonic acid moves from the lipid membrane to the enzyme active site via hydrophobic channel

• Prostaglandin H2 promotes inflammation and gastric acid secretions

How do drugs like aspirin/ibuprofen combat prostaglandin H2 synthase-1 activity?

They donate an acetyl group to Ser 350, which blocks the hydrophobic channel

Describe the potassium ion channel

The potassium ion channel transports potassium ions specifically and quickly. The channel is wider at the cell interior and narrows for the selectivity filter.

• Ions with a radius larger than 1.5 Å cannot pass through.

• Four binding sites within the channel allow for repulsion and flow of ions through the channel.

Sodium ions, with an ionic radius of 0.95 Å, are smaller than potassium ions. But the energy required to lose their solvation shell and pass through is too great, and they do not pass.

Describe: Digestion throughout the body

Mouth

• homogenization to aqueous slurry

• enzymes in saliva: amylase, lipase

Stomach

• denatures proteins with low pH (stimulates secretin)

• denatured proteins better substrates for pepsin (a protease)

Pancreas

• releases NaHCO3 to neutralize acid

• releases digestive enzymes to digest proteins, lipids and carbohydrate

Gall bladder

• releases bile salts required to digest lipids

Define: Protease

Active form. Cleaves proteins

Define: Zymogen

Inactive form. Proteases do not cleave proteins that they are not supposed to cleave – including themselves

Stored in granules near the cell membrane, get released and then activated (usually by cleavage)

True or False: Pepsinogen can self-activate

True

Describe: Digestion of Proteins

• Proteases cleave proteins, zymogens are inactive proteases.

• Many of the proteases are dissolved in the lumen of the intestines

• Peptidases that can cleave oligopeptides are attached to the outside surface of intestinal cells.

• Transporters pass the amino acids and di- and tripeptides into the cells and out into the blood stream

Describe: Digestion of Carbohydrates

α-amylase

• cleaves α-1,4 bonds

• cannot cleave α-1,6 bonds, nor too close

Maltase, α-glucosidase, α-dextrinase

• complete the hydrolysis of starch.

Other carbohydrate-cleaving enzymes are sucrase and lactase, both on the surface of intestinal cells.

Describe: Digestion of Lipids

• Mostly triacylglycerols

• Forms emulsion in the stomach

• Enhanced by bile salts (amphipathic)

• Lipases cleave off two of the fatty acids

• Fatty acids and monoacylglycerols form micelles

• Micelles absorbed across the plasma membrane

What are chylomicrons?

• 2000 Å in diameter

• Transport triacylglycerols, proteins, phospholipids, cholesterol, and fat-soluble vitamins

Define: Metabolism

Metabolism is a series of linked chemical reactions that transforms one molecule to another as required by the organism. This provides required molecules or energy for the organism

Define: Catabolism

Breakdown

fuel molecules → CO 2 + H2 O + useful energy

Define: Anabolism

Building

simple molecules + energy → complex molecules

Define: Amphibolic

Both biosynthetic and degradative

True or False: Degradative and biosynthetic pathways generally occur together

False. Degradative and biosynthetic pathways are generally separate, \n allowing for control.

Glucose is an important metabolic fuel, what is the final product?

CO2

Describe: ATP

• Energy-rich because of the phosphoanhydride bonds

• Can be used to drive other reactions

• Is an immediate donor of free energy in biological systems but is not used for long term storage

Describe: Oxidation-Reduction

Oxidation

• Loss of the electrons

Reduction

• Gain of electrons

The most reduced molecule has the most energy that can be liberated by oxidation. This is why fatty acids have more energy per carbon atom than glucose

Define: Activated Carriers

ATP can be thought of as an “activated carrier” of the phosphoryl group. Other molecules are activated carriers of electrons and two-carbon fragments.

To carry:

1. electrons in fuel oxidation: pyridine nucleotides or flavins e.g. nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FADH2 )

2. electrons in biosynthesis: the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)

3. two-carbon fragments: acetyl-coenzyme A (acetyl-CoA)

Describe: Regulation of Metabolism

Metabolism is regulated through the control of

1. Amounts of enzymes

• how fast they are synthesized • how fast they are degraded

2. Their catalytic activities

• allosteric control e.g. feedback inhibition

• reversible covalent modification

3. the accessibility of substrates

• compartmentalization in eukaryotes

• flux of substrates between compartments

What is energy charge?

Many metabolic reactions are controlled by the energy status of the cell (energy charge)

Most cells have values between 0.80 and 0.95 for energy charge

What coordinates metabolism between different tissues?

Hormones

Describe: Glycolysis

Sequence of reactions converting glucose to two molecules of pyruvate

Occurs in two stages:

1. Trapping and preparation

   • Goal is to trap glucose in the cell and transform into a molecules that can be better broken down (fructose-1,6-biphosphate then glyceraldehyde 3-phosphate)

2. Oxidation to Pyruvate

   • Transfer of phosphate from substrate to ADP (makes ATP)

   • Formation of pyruvate

Outline the steps of Glycolysis

Trapping and Preparation

1. Glucose (Kinase transfers phosphoryl group from ATP to glucose)

2. Glucose 6-Phosphate

3. Fructose 6-Phosphate (Kinase transfers phosphoryl group from ATP)

4. Fructose 1,6-biphosphate

5. Two products formed: Glyceraldehyde 3-phosphate (used in the next step) and Dihydroxyacetone phosphate

Oxidation to pyruvate

6. Glyceraldehyde 3-Phosphate is oxidized and a Pi reduction of NAD+ occurs to form 1,3-Biphosphoglycerate

7. Kinase transfers phosphoryl group from 1,3-BPG to ADP, forms 3-Phosphoglycerate

8. 2-Phosphoglycerate

9. Phosphoenolpyruvate (and H2O)

10. Kinase transfers phosphoryl group from Phosphoenolpyruvate to ADP, forms Pyruvate

What are the major classes of enzymes? How are enzymes labelled accordingly?

1. Oxidoreductases

2. Transferases

3. Hydrolases

4. Lyases

5. Isomerases

6. Ligases

7. Translocases

Labelled EC #.#.#.# where the first number corresponds to the type of enzyme above

Describe: Alcoholic Fermentation

Anaerobic fermentation of pyruvate to ethanol. (Acetaldehyde intermediate)

Describe: Lactic Acid Fermentation

Anaerobic fermentation of pyruvate to lactate (later protonated to lactic acid)

Describe: Regulation of Glycolysis

• Occurs at hexokinase, phosphofructokinase, and pyruvate kinase steps

• All three steps are IRREVERSIBLE

• Tissue Specific

• Controlled by allosteric effectors or covalent modification

Describe: Regulation of Phosphofructokinase

• Most Important Regulation

• Allosteric inhibition by ATP

• Inhibition can be reversed by AMP

• 1st committed step

Step: Fructose 6-phosphate to Fructose 1,6-biphosphate

Describe: Regulation of Hexokinase

• Product inhibition by Glucose 6-phosphate

• Glucose 6-phosphate is not solely used for glycolysis

Step: Glucose to Glucose 6-phosphate

Describe: Regulation of Pyruvate Kinase

• Energy Charge of Cell

• ATP and Pyruvate are made

• Allosteric Inhibition by ATP

• Activation by Fructose 1,6-Biphosphate

Step: Phosphoenolpyruvate to Pyruvate

Describe: Regulation in Muscle

Phosphofructokinase and Hexokinase

Describe: Regulation of Phosphofructokinase in the Liver

• ATP dependent (PH is not important like in muscle)

• Citrate is a key building block

• Fructose 2,6-biphosphate (feedforward stimulation)

• Stimulated when glucose is high

Describe: Regulation of Hexokinase in the Liver

• Glucose 6-phosphate used in liver to synthesize glycogen and fatty acids

• Mainly carried out by hexokinase IV

  • Isozyme

  • No product inhibition

  • Km is 50X greater than Hexokinase

Define: Isozyme

• Catalyze same reaction

• Different kinetics or regulation

• Different primary structure

Describe: Regulation of Pyruvate Kinase in the Liver

• Several isozymes involved

  • L form in liver

  • M form in muscle and brain

• L form

  • Allosteric inhibition by ATP and Alanine

  • Inhibition by reversible phosphorylation

Describe: Regulation by Substrate Availability

Glut 1 *and 3 are always on (Basal Glucose uptake)

Glut 2 only on when Glu is high (In pancreas, insulin regulation)

Glut 4 amount in PM increases with high endurance training

Glut 5 (Fructose transporter)

Describe: Gluconeogenesis

• Conversion from pyruvate to glucose

• Other molecules join when converted to intermediates.

  • Lactate from lactic acid fermentation in muscles

  • Amino acids from proteins

  • Glycerol from triacylglycerols

• Occurs mainly in the liver

Outline the steps of Glucogenesis

Pyruvate

1. Pyruvate carboxylase transforms pyruvate to oxaloacetate

2. Phosphoenolpyruvate carboxylkinase transforms oxaloacetate to phosphoenolpyruvate

3. Enolase transforms phosphoenolpyruvate to 2-phosphoglycerate

4. Phosphoglycerate mutase transforms 2-phosphoglycerate to 3-phosphoglycerate

5. Phosphoglycerate kinase transforms 3-phosphoglycerate into 1,3-Biphosphoglycerate

6. Glyceraldehyde 3-phosphate dehydrogenase transforms glyceraldehyde 3-phosphate to Glyceraldehyde 3-phosphate (also produces glycerol: dihydroxyacetone phosphate)

7. Aldolase transforms glyceraldehyde 3-phosphate to fructose 1,6-biphosphate

8. Fructose 1,6-biphosphotase + Water makes Fructose 6-phosphate + Phosphate

9. Phosphoglucose isomerase makes glucose 6-phosphate

10. Glucose 6-phosphotase + Water makes Glucose + Phosphate

What are the differences between Gluconeogenesis and Glycolysis?

Pyruvate to phosphoenolpyruvate is 2 steps in gluconeogenesis and 1 step in glycolysis

Use of Phosphatases to REMOVE phosphate groups in Gluconeogenesis:

• Fructose 1,6 phosphate to fructose 6-phosphate (Fructose 1,6-biphosphotase)

• Glucose 6-phosphate to glucose (Glucose 6-phosphotase)

Gluconeogenesis USES 4 ATP and 2 GTP, Glycolysis PRODUCES 2 ATP

Describe: Regulation of Gluconeogenesis and Glycolysis

Fructose 1,6-biphosphotase is key.

• Reciprocal to phosphofructokinase

( also Citrate and Acetyl-CoA from TCA and H+ from hydrolysis of ATP)

Fructose 1,6-biphosphotase is inhibited by fructose 2,6-biphosphate (formation catalyzed by phosphofructokinase)

Describe: Synthesis and Degradation of Fructose 2,6-bisphosphate

Glucagon stimulated PKA when blood glucose is scarce. FBPase2 is activated. Glycolysis is inhibited, gluconeogenesis is stimulated.

High levels of fructose 6-phosphate stimulate phosphoprotein phosphatase. Glycolysis is stimulated, and gluconeogenesis is inhibited.

*Both controlled by a single Serine residue (phosphoserine)

Where does gluconeogenesis end in most tissues?

At glucose 6-phosphate. Conversion to glucose occurs mostly in the liver

Can both Gluconeogenesis and Glycolysis occur at once?

No. One pathway is active while the other is inactive.

Describe the transformation of pyruvate under anaerobic and aerobic conditions.

Under anaerobic conditions: Conversion to lactic acid or ethanol (depending on organism)

Under aerobic conditions: Conversion into acetyl CoA, which enters the TCA

What is Acetyl-CoA?

Activated carrier of two-carbon units in TCA

What reaction links glycolysis and TCA? Is it reversible or irreversible? Where does it occur?

The bridge reaction. Irreversible. Occurs in the mitochondrial matrix.

Describe: Pyruvate Dehydrogenase Complex

• Produced from a reaction with pyruvate, CoA, and NAD+

• Consists of Pyruvate dehydrogenase (E1) component, Dihydrolipoyl transacetylase (E2), and Dihydrolipoyl dehydrogenase (E3)

• 8 E2 timers (24 total) forms the core.

• 6 a/B dimers (12 total) are on the face of the cube (2 on each face)

• 6 (a2/B2) dimers (24 total) line the edges of the cube (2 on each edge)

Outline the Steps of Acetyl CoA formation catalyzed by Pyruvate Dehydrogenase Complex

1. Decarboxylation (step 1): rate limiting step, catalyzed by E1

2. Oxidation (steps 2/3): catalyzed by E1

3. Formation of acetyl CoA (step 4): catalyzed by E2

4. Lipoamide regeneration (step 5): catalyzed by E3

Describe the movement/role of lipoamide with the pyruvate dehydrogenase complex

• Lipoamide moves from one active site to another. (E2 surface to E1 to E2 to E3, back to E2 surface)

• Substrate cannot diffuse away.

• High local concentration of substrate.

• Minimizes side reactions.

• Increases rate of overall reaction.

Once Acetyl CoA forms, what occurs?

Glucose CANNOT be regenerated. Acetyl CoA goes on to oxidation by TCA cycle or incorporation in lipids for lipid synthesis.

Describe: Regulation of Pyruvate Dehydrogenase Complex

Allosteric on E2 and E3 (acetyl CoA and NADH)

Covalent Phosphorylation on E1

Describe the differences in the pyruvate dehydrogenase complex at high and low energies

High energy: ATP, NADH, acetyl CoA All inhibit PDH By stimulating kinase

Low energy: ADP and pyruvate inhibit kinase and Ca2+ and hormones stimulate Phosphatase

Describe: Clinical disruptions of pyruvate metabolism

TPP deficiency - low content in rice, more problems with white and polished rice

Beriberi: neurological and cardiovascular disorder (cause by a limited intake of food, malnutrition, famine - thiamine deficiency)

Metal Toxicity (Arsenic/Mercury): high affinity for SH groups in close proximity blocks the reaction

Provide an overview of the TCA cycle:

• Cycle: acetyl-CoA reacts with a product of the previous cycle

• Two carbons go to CO2

• One ATP from ADP and Pi

• Eight electrons – Most of the energy from oxidation is carried by these electrons.

Two stages:

1. Oxidizes two carbon atoms; gathers energy-rich electrons

2. Regenerates oxaloacetate; makes one ATP; gathers energy-rich electrons in NADH and FADH2

Each reaction of TCA is catalyzed

Outline the reactions of the TCA cycle

First Stage:

1. Catalyzed by Citrate synthase to form Citrate (condensation of Oxaloacetate then Citryl CoA hydrolysis)

2. Catalyzed by Aconitase to form Isocitrate (dehydration of citrate, then hydration of cis-aconitase)

3. Catalyzed by Isocitrate dehydrogenase to form α-Ketoglutarate (oxidation of isocitrate, formation of NADH, then decarboxylation of oxalosuccinate)

4. Catalyzed by α-ketoglutarate dehydrogenase complex to form Succinyl CoA, CO2 and NADH. (oxidation of α-ketoglutarate, then decarboxylation)

First Stage Complete, C4 molecules formed

Second Stage:

5. Catalyzed by Succinyl-CoA synthetase to form Succinate (Substrate-level phosphorylation to form ATP or GTP)

6. Catalyzed by Succinate dehydrogenase to form Fumarate (oxidation of succinate, forms FADH2)

7. Catalyzed by Fumarase to form Malate (Hydration of fumarate)

8. Catalyzed by Malate dehydrogenase to form Oxaloacetate (oxidation of malate, forms NADH + H+)

3 NADH, 1 FADH2, and 1 ATP are formed. 2 H2O are used, 2 CO2 are released.

Describe the specificity/Induced fit of citrate synthase in the first reaction of TCA

Sequential reaction: Oxaloacetate first, then acetyl-CoA

Changes conformation upon binding to each, first induced fit, then a preference over hydrolysis of acetyl-CoA. After cleavage CoA leaves which prevents wasteful cleavage of acetyl-CoA.

Does the TCA cycle occur under anerobic, aerobic, or both conditions?

STRICTLY AEROBIC! NADH and FADH2 are not regenerated without oxygen.

Describe: Regulation of the TCA Cycle

• May need to replenish oxaloacetate (e.g. during exercise)

• Pyruvate carboxylase also for gluconeogenesis

  • Requires acetyl-CoA

  • Regulated by energy charge

    • High – make glucose

    • Low – run TCA cycle

• Anaplerotic reaction – “fills up” or replenishes pathway

Within the cycle allosteric enzymes: Isocitrate dehydrogenase and α-Ketoglutarate dehydrogenase regulate:

• α-Ketoglutarate dehydrogenase catalyzes the rate determining step

• Inhibition of isocitrate dehydrogenase leads to increased citrate which inhibits phosphofructokinase and glycolysis (INTERACTION BETWEEN PATHWAYS!)

Does the oxidized form of a substance have a lower or higher affinity for electrons than protons (H2)?

The oxidized form of a substance has a LOWER affinity for electrons than protons.

Does a strong reducing agent (like NADH) accept or donate electrons?

Strong reducing agents DONATE electrons

Does a strong reducing agent (like O2) accept or donate electrons?

Strong oxidizing agents ACCEPT electrons

What does negative reduction potential mean?

What does positive reduction potential mean?

What is the free energy difference from NADH to O2 (electron transport chain)

220 kJ/mol (enough to make 7 ATP from ADP)

What is the electron transport chain?

Electrons exchanged through groups in the active sites of enzymes to produce O2

1. NADH-Q oxidoreductase (COMPLEX 1) /Succinate-Q reductase (FADH2) (COMPLEX 2)

2. Ubiquinone (Q)

3. Q-cytochrome C oxidoreductase (COMPLEX 3)

4. Cytochrome C

5. Cytochrome C oxidase (COMPLEX 4)

What is the role of iron in the electron transport chain?

Iron in the electron transport chain is found in two forms: iron sulfur clusters (in complexes I and II, NADH-Q reductase and Succinate-Q reductase), and as part of heme in cytochromes

Iron goes from ferric (Fe3+) to ferrous (Fe2+) thus reduction potential depends on environment and iron can be involved in many steps

What is the role of copper in the electron transport chain?

Copper goes from Cu2+ to Cu+ in cytochrome c oxidase

What is the role of Coenzyme Q in the electron transport chain?

Coenzyme Q (Ubiquinone) accepts electrons from complexes 1/2 (NADH-Q reductase and Succinate-Q reductase)

• Hydrophobic

• Carries protons and electrons

Describe: Proton Pumps in the Electron Transport Chain

• 3 proton pumps: Complex 1 (NADH-Q-oxidoreductase), Complex 2 (Q-cytochrome C oxidoreductase), Complex 4 (Cytochrome C oxidase)

• Pump electrons across the inner mitochondrial membrane from the matrix to the intermembrane space

What is the link between the electron transport chain and the TCA cycle?

Complex 2 contains succinate dehydrogenase as an integral protein in inner mitochondrial membrane (all other TCA enzymes are in the matrix)

Oxidative phosphorylation (e- transport chain) occurs in the inner mitochondrial membrane

Describe the structure of mitochondria

Inner Membrane: Tightly controlled, impermeable

Outer Membrane: Many channels, voltage dependent anion channels

Cristae: Increase in surface area

Define: Proton Motive Force (Δp)

• Link between electron transport and ATP synthesis provided by a H+ gradient (chemiosmotic hypothesis) Δp = chemical gradient and charge gradient

• TCA in matrix, provides e- ; electron transport chain in the membrane creates H+ gradient to the intermembrane space which is used to make ATP when flowing back

Define: ATP Synthase

T=tight, L=loose, O=open

1: T makes ATP but does not release

2: after rotation T becomes O

3: it now releases ATP

4: ADP and Pi can enter

5: L state traps substrates for next turn

Describe: NAD+ for Glycolysis

• Glycolysis in cytoplasm, oxidative phosphorylation in mitochondria

• Membrane impermeable to NAD+ (or NADH)

• Shuttles e.g. regenerate NAD+

• Yield is only 1.5 ATP (not the 2.5 ATP from NADH) because shuttle uses FAD.

• Runs against NADH concentration gradient

Important in muscle to sustain high rate of oxidative phosphorylation