Metabolic Biochemistry Notes

Metabolic Biochemistry NotesLecture 1-Introduction

1. Enzymes

2. bioenergetics

3. metabolic pathways

4. compartmentalization of pathways

5. metabolic adaptations:

a. diabetes

b. endurance sports

c. starvation

6. metabolic disease

7. enzyme analysis

Metabolism- Is the complex combination of physical and chemical processes occurring in a living cell or organism. It is broken down in two main processes:

1. Catabolism- processes that break down large molecules to yield energy Eg glycolysis- breakdown of glucose

2. Anabolic synthesis of compounds needed by the cell Eg. Lipids, amino acids

Sources of Energy:1. Carbohydrates such as (potatoes bread rice)- Starch- Glucose-ATP

2. Protiens such as meat fish eggs- Amino Acids and Nitrogen- ATP

3. Fats and Lipids (animal products)- Fatty acids and Acetyl CoA-ATP

ATP-Adenosine Triphosphate is the energy currency of ALL cells. And is a ribonucleuotide containing 3 phosphate groups. Energy stored in the phosphate bonds when one phosphate group is donated 30.5 kJmol-1 of energy to produce ADPMetabolic Pathways are a series of reactions that are normally catalyzed by enzymes. Reactants and products share intermediate products. Exergonic energy is normally produced through the breakdown of metabolites through catabolic reactions.

Types of pathways:A,B and C linear pathways

D cyclic pathway

Reduced Molecules release more energy ( contain more hydrogen) the more it becomes oxidized ( more Oxygen on the molecule)less energy it produces.

Example: Methane -196 Kcal mol-1

Carbon dioxide 0 Kcal mol-1Inherited Metabolic Diseases- Majority is due to defects of single genes that code for enzymes that facilitate conversions of various substances (substrates) into others (products). Example albinism is due to the deletion of tyrosinase enzyme that produces the melanin in pigment in hair skin and eyes.

Metabolomics- cell profiling were cellular activity is determined due to chemical processes the cell undergoes to determine defects and damages may occur compared to healthy cellular activities.

Lecture 2- Bioenergetics Metabolism and Enzyme Catalysis

Metabolic Pathways-

Catabolism breakdown is the breakdown of large products into smaller more usable products.

Anabolism is the build up of the catabolic products to make larger and newer productsLaws of Thermodynamics:Principles that describe the flow and exchange of heat, energy, and matter

Deals with systems at equilibrium

Determine if a system is stable or if a reaction can occur spontaneously

Systems are:

Isolated : No exchange of either energy or matter

Closed : No exchange of matter, may exchange energy

Open: May exchange energy or matter (living systems) HUMANS!!

Equilibrium:

Le Chatliers Principle- A system at equilibrium will change in order to absorb anything that has occurred from the outside.

The human body is constantly counteracting effects of the outside environment and internal environment through food and fluid to keep equilibrium constant and at homeostasis.

Equilibrium constant= Products/ Reactants Equilibrium constant Keq = [C][D]/ [A][B]

CD- concentration of products

AB- Concentration of reactants

Keq greater than 1 the reaction will tend to the right (direction of products)First law of thermodynamics: Conservation of energy

Energy cannot be created nor destroyed but it can be created or transferred. But the TOTAL energy of the system does not change

Second Law of Thermodynamics: The universe tends towards increasing disorderEntropy is the measure of disorder.Human system is highly ordered, we do not break the second law because we decompose to simple matter. We release heat which generates more disorder.

Free Energy- Gibbs free energy G- this energy is free to do work meaning energy that is broken between C-H bonds in molecules to create energy (ATP). G- Change in Gibbs free energy.+G- Unfavorable reaction (energy has to be supplied in order for reaction to take place)

-G- favorable reaction (Reaction happens with no excess energy supplied to reaction)

Entropy is regarded as favorable reaction as it tends to go relates to disorder which uses free energy and doesnt require any energy from an outside source.Examples:

+G- making bacterial cell wall, made of peptidoglycan made of repeating units of NAM and NAG, building blocks that fits together via bridge to make the cell wall. Disordered state (NAM and NAG separate blocks) to and ordered state ( bacterial cell wall) against entropy

-G- Is the degradation of glycogen to glycogen phosphorylase (GP). Glycogen string of glycogen units broken down into individual molecules of (GP) with entropy.Energy lost to entropy is unusable energy which is mainly heat.

Useable energy- chemical energy can be used to breakdown molecules and do work.

Useable energy + non useable energy = Total energy

Exergonic reactions have a -G ( products have less energy)

Endergonic reactions have a +G (energy of the products is higher than the reactants)

Terminology:

G- Change in free energy

H- Change in enthalpy (heat)

S-Change in entropy

T- Temperature (Kelvins)

G= H- TS

G is the driving force towards equilibriumGo (Go) primeStandard transformed constants

Standard biological conditions (buffered solutions)

[H+] = 10-7 M (pH 7)

[H2O] = 55.5M

G 'o = -RTlnK ' eq G 'o- standard biological condition

R- Gas constant

T- Temperature in Kelvin

Keq- products/ reactants

1n- inverse log10When Then And K ' eq > 1.0 G 'o = -ve reaction proceeds forwards

K ' eq = 1.0 G 'o = 0 reaction is at equilibrium

K ' eq < 1.0 G 'o = +ve reaction proceeds reverse Reaction Coupling: Taking an unfavorable reaction with a favorable reaction to power the unfavorable reaction in order for it to become favorable overall.

Example glycolysis:

Reaction 1 Endergonic- Glucose + Pi (inorganic Phosphate PO4) Glucose 6- phosphate (has more energy than reactants)Reaction 2 Exergonic- ATP ADP + Pi (Pi is a product or reaction 2 and a reactant of product 1, therefore putting both reactions can be put together to get a favorable reaction!!!

Reaction 3 Exergonic- Glucose+ ATP Glucose 6 phosphate + ADP Free energy from reaction 2 is powering reaction 1.

Oxidation is the loss of an electron Reduction is the gain of an electron Oxidation-reduction reactions always occur together (coupled).

Succinate donates electrons and becomes oxidized

FAD accepts electrons and becomes reducedATP- energy currency of cells

ATP-ADP releases 30kJmol-1 of energy

ATP- is continuously made and broken down in the body

Oxidation and Reduction reaction is the transfer of electrons. Highly reactive and cannot float around freely as free radicals that cause problems. Use electron carriers to hold the securely and transfer them some were else were they are needed in the body.An example of this is NAD- Nicotinamide Adenine Dinucleotide (vitamin B3 niacin).Goes from an oxidized form and reduced form ( picks up 2 electrons and 2 protons( H atom that lost its electron))

NAD- Oxidised form electron acceptor

NADH- reduced form electron donor

NAD coenzyme- organic molecule that is free in the cytoplasm and involved in enzyme function.

NADH carriers electrons to the transport chain, electrons go down electron transport chain to make ATP Carbon dioxide and water. Within electron transport chain the Oxygen is known as the terminal electron acceptor.

NADH- electron energyATP- breaking bonds to produce energy

Both forms provide energy for the cell.

THE MORE REDUCED THE COMPOUND IS THE MORE C-H BONDS THE MORE ENERGY THERE IS!!!!!

More oxygen less energy produced eg Methane CH4 high energy, CO2 no energy

Use fat as an energy store opposed to glucose because glucose needs water to react, fat removes water. Fat is highly reduced. Glucose is less reduced but easier to metabolize.

Lecture 3- Enzymes: Kinetics and Regulation

Enzymes:

Enzymes enable molecules called substrates to undergo a chemical change to form new substances called products. Each enzyme acts on a specific molecule or group of molecules called substrates. Each substrate fits into an area of the enzyme called the active site, the enzyme changes shape a little with the substrate. In he enzyme substrate complex, the enzyme holds the substrate in a position were the reaction can occur easily, after the reaction releases the products and carries out the same reaction again and again. Catalysts- Catalyze is a biological enzyme that breaks down peroxide bubbles in the liver, peroxide breaks down into water and CO2 but is very slow add FeCl2 as an inorganic catalyst and reaction runs 1000X faster. However add catalyze breaks down peroxide in a biologically relative timeframe which is 140 million reactions every second.

Almost all enzymes are proteins. Highly specific to one substrate and one reaction only do not do random things.

Reduce the activation energy of a reaction.

DO NOT alter equilibrium position

Recognize substrates with high specificity and sensitivity. Dont need much reactant in cytoplasm in order the enzyme to find it and do its job.

Enzymes dont alter equilibrium constant

ALL enzymes will work with equilibrium and based on le chateliers principle

Activation energy- All reactions go through an intermediate state which is a high energy molecule. The activation energy is the energy required to get over this intermediate state. The higher the activation energy ( transition state ) the slower the reaction and is due to same charges being to close together, or molecular bonds being held together that is unfavorable, this is a high energy state. In order to get over the high energy state enzymes stabilize the transition state bring reactants closer together and control repelling charges and stabilize high energy bonds and overall reducing the activation energy and increasing the rate of the reaction. Multiple enzymes can speed up reactions to a relative timeframe example Neurons can have multiple overlapping reactions in order to generate an impulse. DOES NOT CHANGE THE G Enzyme and substrate have an induced fit were the enzyme fits in the substrate to change its electrochemical charge and shape to make sure its a perfect fit. A specific enzyme will fit into a specific substrate and bind to its active site. HIGHLY SPECIFIC!! Enzyme + Substrate ES EP E + Product ALL enzymes are stereospecific enantomers and chiralClassification of EnzymesEnzymes are classified based on there enzymatic function.6 Classes:

Oxidoreductases catalyze oxidation-reduction reactions BH2 + A B + AH2Transferases catalyze transfer of functional groups from one molecule to another D-B + A-H D-H + A-BHydrolases catalyze hydrolytic cleavage A-B + H2O A-H + B-OH Ligases and Synthetases Bond formation (reverse of hydrolase) coupled to ATP hydrolysis Lyases catalyze removal of a group from or addition of a group to a double bond or other cleavages involving electron rearrangement A-B A + B [ synthases: A + B A-B ] Isomerases catalyze intramolecular rearrangement R-A-B A-B-RThe theory behind the substrate fitting into the enzyme active site is not accurate as the enzyme catalytic site might not be the perfect fit for the substrate, so some of it that is in the wrong charge or hydrophobicity, but when the substrate approaches the enzyme at a close proximity there is molecular movement to get that that perfect fit. THIS IS INDUCED FIT!! However substrates that have a slightly different shape CANNOT INDUCE FIT. This is what gives us enzyme specificity.

ONE SUBSTRATE ONE ENZYME ONE REACTIONWrong shaped substrate Substrate binds to enzyme (conformational change) Perfect match

Non Substrates No conformational change

Substrate enzyme have a slightly different shape as they approach each other they change shape to allow perfect fit which ultimately changes the substrate and enzyme. BOTH CAN HAVE CONFORMATIONAL CHANGE!!

Alcohol interacts with neurotransmitter channel called GABA to slow it down. Alcohol changes the sodium and potassium pump in the neuron.

What causes Hangovers?

Kinetics:

Alcohol dehydrogenase (ADH)- When alcohol reacts with the enzyme produces acetylaldehyde which is toxic, this is an oxidation reduction reaction.

Technically all reactions are reversible, and all go until equilibrium is reached.

Even if there is a decrease in free energy it has to go over the activation energy which is were looking at an intermediate (enzyme substrate intermediate) that has unfavorable bonds or charges. The enzyme decreases the activation energy by stabilizing enzyme substrate intermediate.Rate:

Rate is the amount substrate converted to product per unit time. Rate is related to a rate constant, so the rate of reaction is related to the decrease of reactants = rate constant and concentration of products. Rate constant goes in TWO DIRECTIONS- forward reaction= reverse reaction. This is for a simple react - (not negative value, decrease value)[A] = k [C] decrease in [A]

[C] = k [A] increase in [C]Rate reaction reactants X concentration of products

Enzyme + Substrate ES EP E + ProductES- substrate intermediateWe can calculate the rate or velocity of the reverse and forward reactions. Rate constant of forward reaction K+1- lots of reactants (A) little product (C)Rate constant of reverse reaction K-1-little reactants (A) lots of products(C)

Rate constant at equilibrium K0- equal amount of products and reactants A=C

Enzyme Recycling

Enzyme + Substrate ES EP E + ProductE+S- reversible reaction to ES intermediate

ES- reversible reaction (catalysis has occurred) with EP intermediate

EP- reversible reaction of E+P- enzyme is recycled

Lag phase happens in less than a millisecond was the enzyme is finding the substrate.

Theres a direct linear relationship between product and time. Also known as steady state, were the ES concentration looks constant. Steady state kinetics is where the reaction E+SESP happens so quickly it looks constant.

Rate equation: Rate= rate constant X substrate concentrationAt equilibrium the rate of forward reaction = rate of reverse reaction.

K+1=K-1

K+1/K-1= concentration of products/ conc of reactants = Keq( equ constant)

Steady state were the ES intermediate remains constant. The very initial parts of the reaction ( ignore reverse reaction) therefore ES is constant.

Limiting rate factor is the catalysis between E+SES.

V0 = Vmax [S]/(Km + [S]) THIS IS THE MICHAELIS-MENTEN EQUATION !!!!

V0- initial velocity (rate)

Vmax- maximum velocity

[s]- substrate concentration

Km- michaelis- menten constant

Plot- initial velocity V0 vs. Substrate concentration

When this plotted eventually Vmax will be approached, but is an asymptote were the line goes forever and will never reach Vmax value. This is an estimated reading.

Km is defined as substrate conc that gives half Vmax both can be estimated by the graph.

Vmax is the point that where increasing the substrate conc does NOT increase rate. This signifies how fast the enzyme can go and its estimation only via the asymptote.The higher the Km the greater the amount of substrate needed to half Vmax.

>Km > [S]Vmax

Km- can be a measure of affinity between enzyme and substrate, lower Km indicates higher affinity between enzyme and substrate binding rate.

Higher the Km the lower affinity and slower binding rate.

Very initial part of the graph we start with a low [S] and slow rate, as we increase the [S] we increase rate and eventually gets to a time were the enzyme is saturated, the limiting factor is in the binding and catalysis of the enzyme. The more substrate will not increase enzyme productivity. THIS IS VMAX

Mitochondria has a low Km therefore has more affinity to bind substrates (acetaldehyde) and have a faster rate of reactions. With the high Km in the cytosol it takes longer

( increased amount of acetaldehyde to reach half Vmax) In mitochondria there is a low amount of acetaldehyde. Genetic defects in the enzyme within the mitochondria means that the enzyme will not work as well and relies on the cytosol enzyme to remove the acetaldehyde (toxic). This builds up quicker in the body and causes a hangover.

Lineweaver- Burk Plot- allows you to calculate 1/ vmax and -kmIs a linear plot y=mx+b1/V0= km/vmax X 1/[S] +1/vmax

Questions

1. The induced fit model of enzyme and substrate interaction involves:

a)Enzyme and substrate fitting exactly and no molecular movement required for binding

b)Enzyme first reacting with substrate in order to induce fit

c)Enzyme and substrate not fitting exactly and some molecular movement required for binding

d)Enzyme not interacting with substrate as it does not partake in the reaction

2. Enzyme steady state kinetics is when:

a)The reaction is at equilibrium

b)No further product is being made

c)Substrate is being consumed at maximal rate

d)The enzyme-substrate concentration appears constant 3. The Michaelis-Menten plot is a)A graph that compares substrate concentration to reaction rate

b)A graph from which maximal reaction rate can be estimated

c)A graph from which the Michaelis-Menten constant can be determined

d)All of the above

e)None of the above

4.The Lineweaver-Burk plot is a)A graph that shows the relationship between the inverse of substrate concentration and reaction rate

b)A graph from which maximal reaction rate can be calculated

c)A graph from which the Michaelis-Menten constant can be calculated

d)A graph which can be used to investigate enzyme-inhibitor interaction

e)All of the above

Enzyme Inhibitors:There are 2 types of enzyme inhibitors reversible and irreversible.

1. Irreversible are those under biological conditions cannot be reversed. They dissociate very slowly from the target.

2. Reversible are used in metabolism, almost everything in our body is regulated to keep homeostasis and has rapid dissociation, can turn on and off many times a second. 3 forms enzyme inhibitors: competitive, uncompetitive and mixed (uncompetitive).

Irreversible inhibitors:

Penicillin- prevents the cell wall extension and replication (NAM and NAG) cell wall is made peptidoglycan, polymer of NAM and NAG and linked together by a peptide cross bridge, which is done by transpeptidase , -lactans stop the transpeptidase from acting and therefore cell wall cannot be made stopping the bacteria from dividing, therefore penicillin inhibits bacterial cell division. It acts on the - lactam ring. The bacteria produced -lactamase- an enzyme that opens the - lactam ring to open the ring and stops the penicillin from working. Clavulanic acid is used to destroy -lactamase and has been mixed with penicillin so it can take its original effect.

Competitive inhibitors

Bind to the enzyme catalytic site to become an enzyme inhibitor complex, they compete with the substrate for the catalytic site, when the enzyme and inhibitor are bound there is no catalysis. Inhibitor is similar to the substrate in shape charge and hydrophobicity, and it binds to the active site of free enzyme. The substrate and inhibitor bind to the same site therefore if you add more substrate it out competes the inhibitor and reaction occurs normally. Used in normal metabolism and drug development.Uncompetitive Inhibitors

ONLY bind to the substrate enzyme intermediate, they bind to another site NOT CATALYTIC SITE. When ES bind opens up another site for the inhibitor to bind. A free enzyme does not have this site when ES bind it opens up this site. ONLY BINDS TO ES COMPLEX.

Mixed inhibitors

Combine to the enzyme and ES intermediate and create another site on the enzyme. NOT the catalytic site. Inhibitor binding site is present in the enzyme alone and ES intermediate. Binding at another site, increasing the substrate concention will have no affect as its binding to other site then what the substrate is binding.Enzyme Regulation

Feedback control- Allosteric modulation is a mixed or uncompetitive inhibitor binding to a site that is NOT the catalytic site. Is Negative feedback control were the product binds to the initial enzyme and stops the chain reaction. This is a conformational change. The inhibitor DOES NOT fit in the catalytic site so it binds somewhere else to get a conformational change in the active site and substrate cannot bind

Allosteric modulation happens at the beginning and branch points of a reaction, so we dont use up substrate and make too much products that are not needed.Allosteric Modulators:

Can be positive or negative and proteins (enzymes) can go between the Taut state (less active) and relaxed state ( more active)

Positive effector tautrelaxed more active

Negative effector relaxed taut less active

Produce a sigmoidal shaped graph with velocity Vs Substrate.

Controlling Enzymes

Cleavage- not all are produced in active forms some are produced as zymogens.

Zymogens are inactive form of the enzyme thats released in the blood and is longer structure that has to be cleaved in order to become active.. Trypsinogen is an enzyme that is cleaved by pepsinogen in the stomach to make trypsin. Clotting factors they need to be turned on and off at certain times, it is turned on by a enzyme cascade and starts initially as a zymogen that is activated which acts on another enzyme to activate it and so on.

Phosphorylation system can turn on and off - Kinase phosphorylates and phosphatase dephosphorylates. Classic system of a phosphorylation is the transduction pathway from outside the cell to inside the cell.

Questions

1. A competitive inhibitor can a)Bind to an enzyme active site

b)Be part of allosteric modification

c)Be outcompeted by substrate

d)(a) and (c) only

e)All of the above

2. Feedback control can a)Only work in a negative fashion

b)Only work in a positive fashion

c)Work in a negative and positive fashion

d)Work only in complex, multi-subunit, multi-step enzyme pathways

3. In enzyme catalysis, allosteric modulation is when an effector binds to a site that is not the active (catalytic) site and alters the shape of the enzymeFALSE4. In enzyme control, covalent modifications include phosphorylation carried out by enzymes called phosphatasesFALSELecture 4- Glycolysis and GluconegenesisGlycolysis is the breaking of 6 carbon chain to two 3 carbon chains (from glycose, an older term[1] for glucose + -lysis degradation) is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO + H+. The free energy released in this process is used to form the high-energy compounds ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide). There are 10 steps involved in this process. We dont store glucose because it needs water to maintain solubility while fat doesnt need water. The GOAL of Respiration- is taking C-C and C-H bonds and oxidizing them to produce energy. Oxidation is loss of electrons to produce energy, end point ATP, intermediate electron carriers NADH (power the production of ATP).Carbon dioxide produces the most energy as a product from the reduction of pyruvate as it has no C-H bonds and has been completely oxidized. Preparatory Phase is the phosphorylation of glucose to glyceraldehyde 3-phosphate (GAP) steps 1-5:

1. Priming action phosphorylation of glucose, traps glucose in the cell and need high energy phosphate groups to release energy and binding of phosphate groups to the enzyme lowers activation energy :

a) Glucose Glucose 6- Phosphate, using Hexokinase enzymeb) ATP ADP need energy to start the reaction.c) FORWARD REACTION

d) Enzyme glucokinase aka hexokinase

2. Isomerization :

a) Phosphoglucose isomerase enzyme turns glucose 6- phosphate to fructose 6 phosphate in catalytic reaction.

b) Reversible reaction3. Seconding Priming action:

a) ATPADP

b) Fructose 6 Phosphate Fructose 1,6- Biphosphate (one phosphate group on both 1,6 carbons on ring), using the enzyme Fructophosphokinase

c) FORWARD REACTION

4. Cleavage:a) Aldolase enzyme BREAKS the Fructose 1,6- Phosphate in two separate 3 carbon chain molecules:

dihydroxyacetone phosphate (DHAP) glyceraldehyde 3-phosphate (GAP 1)

b) Reversible reaction

5. (DHAP) is not useful so the Triose Phosphate isomerase enzyme converts it to (GAP 2)

a) DHAP GAP2 using triose phosphate isomerase enzyme.

Pay off Phase: Oxidative conversion of GAP to pyruvate and coupled ATP and NADH (electron acceptor) (ENERGY)

6. Phosphorylation (of organic phosphate from cell cytoplasm) and Oxidation of BOTH GAP 1+2 molecules:a) GAP 1,3 Biphosphoglycerate using enzyme triose phosphate dehydrogenase

b) 2NAD+NADH+ H+ Oxidised (Electron reaction) conservedc) GAP + 2PO4 high energy phosphate is lost to produce ATP7. First ATP forming reaction (substrate level Phosphorylation) Dephosphorylation:

a) Enzyme Phosphoglyceralkinase catalyzes BOTH phosphates to ADP.

b) GAP 3- phosphoglycerate

c) ADP ATP

d) Reversible reaction8. Enzyme Phosphoglyceratemutase changes position of phosphate group in 3- phosphoglycerate to 2- phosphoglycerate.

a) Reversible reaction

9. 2- phosphoglycerate loses water phosphoeonalpyruvate (PEP) using the enzyme enolase.a) Revisable reaction

b) Very high energy phosphylated compound, transfer it ADP to make ATP

10. The Second ATP forming reaction:

a) Enzyme pyruvate kinase mediates the transfer of phosphate group from 2-phosphoglycerate to an ADP molecule.

b) ADP ATP

c) FORWARD REACTIONSimple Summary of Glycolysis Reaction:Steps 1-3- 6 carbon compound with 2 ATP two phosphate groups Conversion of glucose to fructose 6 phosphateusing a series of enzymes:

a) Hexokinase

b) Phosphohexoisomerase

c) Phosphofructokinase-1

Step 4- Fructose 6 phosphate molecule is cleaved forming 2 X 3 carbon chained moles GAP and DHAP using Alodolose enzyme.

Step 5- DHAP converted to GAP by Triose phosphate isomerase

Step 6- BOTH GAP molecules are Oxidised by triosephosphate dehydrogenase and gain a phosphate group.Step 7- ATP is formed using enzyme phosphoglyceralkinase by transferring the phosphate groups to ADP.Step 8- phosphate group moved from 3C to 2C on phosphoglycerate molecule using phosphoglyceromutase enzyme.Step 9- Dehydration reaction of 2- phosphoglycerate molecule, using enolase enzyme and producing phosphoenolpyruvate PEP (high energy phosphate molecule)Step 10- High energy phosphate group from PEP molecule binds to ADP molecule and produces ATP and pyruvate.

The body needs a constant supply of ATP in order to power cells. Eg charge separation between cells

Glycolysis- produces 2 ATP

Glycolysis overall reaction

Glucose + 2ATP + 2NAD+ + 4ADP + 2Pi

2 pyruvate + 2NADH + 4ATP + 2ADP + 2H+ + 2H2O

Control:

Isozymes- same function but slightly different protein sequence or different tissue locations. They generally have different catalytic activities.

Blood glucose glucokinase enzyme is at Vmax,

The liver does not rely on glycolysis as energy production for itselfGluconeogenesis-

Gluconeogenesis- making glucose from other sources. Mainly in the liver. Does not produce ATP but uses it up. Glucose is the major fuel source for the brain, kidney medulla sperm and red blood cells. Brain uses 120g of glucose a day which is greater than the liver glycogen stores. Synthesis of glucose from pyruvate. 3 reactions that cannot be reversed have high -G, 7 reactions are at equilibrium.

The 3 by pass reactions:

Bypass 1 step 10 of glycolysis

1. Bypass 2 Step 3 of glycolysis reaction

2. 3. Bypass 3 step 1 of glycolysis reaction

The first by pass is reaction 10, Take PEP high phosphate molecule and convert it to pyruvate and generate ATP.

These reactions need different enzymes Step 1 is a kinase so phosphorylation reaction

Step 2- dephosphorylation reaction

Step 10- 2 step reaction because Forward reaction in favorable. REVERSE REACTION has too high G so needs 2 reactions that happens in the mitochondria: Bypass 1- step 10

a) Pyruvate + HCO3- + ATP oxaloacetate + ADP + Pib) oxaloacetate + GTP PEP + GDP + CO2 The mitochondria has an excess of NADH, we dont have an excess in the cytoplasm but we need NADH in the cytoplasm otherwise gluconeogenesis will stop. Therefore pyruvate enters the mitochondria turns gets turned into oxaloacetate, which regenerates NAD in the mitochondria. Oxaloacetate cant leave the mitochondria, so its converted to malate that can transport out. The malate is then regenerated to oxaloacetate to regenerate NADH in the cytoplasm.

All related to creating NADH in the cytoplasm so gluconeogenesis can keep going.

Bypass 2 step 3 in glycolysis reaction

The forward reaction is one phosphate two phosphate requires energy input. Not easily reversedPhosphofructokinase reaction bypassed by a simple hydrolysis reaction:

Phosphofructokinase (Glycolysis) catalyses:

fructose-6-P + ATP fructose-1,6-bisphosphate + ADP

Fructose-1,6-bisphosphatase-1 (Gluconeogenesis) catalyses and chops a phosphate group off:

fructose-1,6-bisphosphate + H2O fructose-6-P + PiBy pass 3- is step 1 of glycolysis.

Hexokinase is only found in the liver and a little bit in the renal medulla. The liver is involved in maintaining blood glucose, if every tissue had the ability to dephosphrylate glucose it will become straight glucose and will leave the cell. No transport system for glucose-6-phosphate

Hexokinase reaction bypassed by a simple hydrolysis reaction:

Hexokinase (Glycolysis) catalyses:

glucose + ATP glucose-6-phosphate + ADP

Glucose-6-Phosphatase (only in the liver) (Gluconeogenesis) catalyses:

glucose-6-phosphate + H2O glucose + PiGlycolysis:

glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP

Gluconeogenesis: 2 pyruvate + 2 NADH + 4 ATP + 2 GTP glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi Gluconeogenesis uses more ATP than what glycolysis produces. Glycolysis produces a net of 2 ATP and gluconeogenesis needs an input of 6 phosphate bonds therefore we are wasting 4 phosphate bonds if these reactions were running in tandem.

Use more energy then we produce When glycolysis in on gluconeogenesis is off and vice versa. This is done by allosteric modulation (turning enzymes on and off) also known as reciprocal regulation.

The 3 bypass reactions have a high -G and are used as the regulatory steps.

Hexokinase 1st step in glycolysis- product is glucose-6 phosphate and gives it negative feedback; glucose-6 phosphate turns hexokinase off. Aka product inhibition. Therefore if there is a lot of glucose in the cell theres no need to keep trapping more so it goes through the negative feedback system.

Glucokinase- is not inhibited by its own product, therefore continues to use glucose to make glycogen. So G6P can continue to be produced in the liver and be made stored as glycogen. Still active at high blood sugar, continues to produce glucose in order to reduce blood sugar level to 4.5mmol. Hexokinase can be controlled on a DNA level, so high glucose and muscle exertion turns hexokinase.

In the liver low blood sugar initiates G6P transcription.

Regulation at the protein level is the regulation of phosphofructokinase-1

Pyruvate kinase is acted upon by ATP and actylcoA to turn glycolysis off because the cell is energy full. However fructose 1,6 biphosphate activates pyruvate kinase. When there are a lot of metabolites present fructose 1.6 phosphate activates the pyruvate kinase enzyme to remove the metabolites. Conversely acetyl CoA turns glycolysis off and turns gluconeogenesis on. This is reciprocal regulation

Pentose Phosphate Pathway- needed to make sugars for DNA and keep NADP reduced. The reducing power is to keep glutathione reduced and NADPH keep fatty acids reduced.

NADP and NADPH are needed for several reactions, in order to make nucleotides and coenzymes we need to be able transfer NADP to NADPH and then these two are necessary for keeping glutathione reduced and NADPH is used in fatty acid synthesis.Lecture 5- Pyruvate Dehydrogenase (PDH) and Citric Acid Cycle (CAC) Does not produce ATP but does produce NADH.

Produce electron carriers that undergo oxidative phosphorylation and produce ATP.

Oxidative respiration- NOTHING TO DO WITH OXYGEN ITS THE REDOX REACTION!!!

OXIDATION- LOSS OF ELECTRONS

REDUCTION- GAIN OF ELECTRONS

Reaction happens in the mitochondria Mitochondria has been theorized to come from endosymbiosis based on its double membrane, circular chromosome and ability to divide by itself. Has an outer and highly folded inner membrane called cristae. Outer membrane is porous allowing substance to cross with no issue while the inner membrane is selective (ion channels).

The inner membrane is IMPERMEABLE TO IONS which is very important, without it oxidative phosphorylation cant happen. The space created by the cristae creates a matrix that the pyruvate eventually enters. The matrix is like the cytoplasm of the mitochondria. In the matrix we have PDH and CAC.

Outer membrane has large channels that are not very selective.

Pyruvate to get into the matrix from the cytoplasm has to go through a symporter. It is a transmembrane protein channel that takes pyruvate and hydrogen.

Matrix needs a lot of hydrogen for oxidative phosphorylation. And pyruvate for CAC.

Since its impermeable to ions the ions can only get through specific protein channels eg hydrogen pyruvate symporter channel.

Energy used muscle contraction- ATP is bound the myosin head cannot bind to actin, ATP is hydrolysed to ADP and phosphate get a conformational change PO4 is released and binds to a new location on actin. ADP is released and we get a conformational change that pulls actin along. ATP then binds and dissociates from actin. Slow twitch fibres- slow oxidative (needs oxygen) rich in blood lots of myoglobin and mitochondria, slow to contract but long lasting eg postural muscles marathons. Very red in colour

Oxidative and glycolytic uses ANAEROBIC glycolysis as well as oxidative CAC and oxidative phosphorylation. Rich in blood, mitochondria and myoglobin (oxygen storing protein ). Pale pinker colour

Fast twitch oxidative- uses ANAEROBIC glycolysis as well as oxidative aerobic glycolysis. This is good for sprinting, short fast bursts. Mostly glycolysis, not a lot of blood, myoglobin and mitochondria. White in colour.

Enzymes- acetyl CoA is the crossroads were many metabolic pathways converge such as fatty acids and proteins. Different substances such as sugar fat and protein are catabolized to produce acetyl CoA, which is then fed into the CAC, it is a major entry point for energy production. Acetyl CoA- cannot go back into glueconeogenesis due to a high G, sugar can go back into fat under physiological conditions.

Under anaerobic conditions pyruvate does not go into PDH, instead pyruvate goes into otherterminal electron acceptors (lactate) bacteria (ethanol).

Pyruvate Dehydrogenase complex: Is a massive complex 3 different enzymes- within the massive complex there are multiple copies of each enzyme. In each complex and have a variety of names. Highly conserved.

Highly exergonic

5 different coenzymes- coenzyme is something that is needed for the enzyme to work, and is regenerated at the end as well as the enzyme. Dont partake in the reaction.

coenzyme Pyruvate AcetylCoA NAD+NADH

FAD, lipoic acid and thiamine pyrophosphate are permanently bound.

CoenzymeA and NAD+ are free to move around the cytoplasm and need to be continuously controlled by a kinase which phosphorylates and a phosphatase that dephosphorylates. Multiple copies of 3 enzymes:

E1 (pyruvate dehydrogenase, 20-30 copies)

E2 (dihydrolipoyl transacetylase, 60 copies)

E3 (dihydrolipoyl dehydrogenase, 6 copies)

5 coenzymes:

CoenzymeA- carries acetyl groups and acetyl are 2 carbon compounds. It is able to pick up substance because of the SH group and form an S group. Used in a lot of biological processes.NAD+ - from niacin vitamin B3 can pick up 2 electrons forming a hydride ion (1 proton and 2 electrons) one of the protons bind to NAD and the other is released to the cytoplasm, have to have H+ in the cytoplasm to act as a buffer.FAD- Vit B2 permanently bound to PDH same as the hydride ion, both NAD and FAD are electron carriers. Both go into REDOX reactions. lipoic acid- lipoamide produced from lipoic acid, can go between the REDOX reactions of lipoamide. Therefore is able to pick up other compounds. Lysine molecule one cofactor compound enables the movement from oxidized to reduced form.thiamine pyrophosphate (TPP)- thiamine vit B1, capable of becoming ionized were the hydrogen ion can dissoaciate and leaves a negatively charged ion and called the carbanion ion. Thiamine diffieciency causes beri beri disease affects the CNS and neural communication is disrupting causing muscle weakness, thiamine is needed for CAC. Regulatory enzymes:

kinase

phosphatase

Overall pyruvate dehydrogenase complex

1. Pyruvate from glycolysis.

2. Need Acetyl CoA

3. Need electron donor NAD+ and acceptor NADH

4. All cofactors TPP, FAD and Lipaote

5. Produce Acetyl CoA goes to the citric acid cycle and CO26. Has a large -G therefore cannot be reversed back into sugar.

Yellow E1

Green E2

Pink E3

E1- pyruvate is decarboxylated and produces carbon dioxide and is released. The 2 carbon compound (acetyl) binds to TPP. E2- S-S group becomes reduced to SH group and the other S from the S-S group binds with the 2 carbon group from E1. This 2 carbon compound is transferred to CoenzymeA to make acetyl CoA- which goes to the CAC, and when that happens we have 2 SH groups so its reduced. However the SH group has to S-S group.

Acetyl CoA

E3- Takes both hydrogens from SH group and transfers them to FAD to make FADH2 and in turn goes back to SS, which is regenerated to what we need on E2. In order for electrons to moved has to be turned into NAD and go into NADH which is mobile not bound like FAD. By doing this FAD is regenerated.

Control of PDH

Lots of ATP turn on pyruvate kinase and the kinase phosphorylates pyruvate dehydrogenase and gets turned off. Same as acetyl CoA if theres fatty acids in the cell

Citric Acid Cycle

Stage 1 steps 1-5- introduction of 2 carbons from acetyl CoA (2 carbons from acetyl and CoA is regenerated to PDH), loss of 2 carbons to CO2 and production of NADH and one GTP, which is converted to ATP.

Stage 2 steps 6-8- partial oxidation succinate and oxaloacetate, make NADH and FADH2 and we regenerate oxaloacetate.

Stage 1 Reaction 1- citrate synthase (makes citrate) is a dimer, shows induced fit really well also has very specific binding mechanism.

Oxaloacetate (from CAC) is 4 carbons and acetyl CoA (from PDH) is 2 carbons and forms citryl CoA that is 6 carbons, which is dehydrated and CoA group is removed and regenerated to form citrate. If acetyl CoA bound first it would be cleaved releasing.Has very specific binding mechanism were the oxaloacetate has to bind first and the acetyl CoA binds second in order for the 2 carbon to bind with the 4 carbon and make the 6 carbon chain, if Acetyl CoA bound first it would be catalytically cleaved releasing the 2C and CoA, therefore the 2C will not enter CAC. This order means we dont lose the 2C group.

Reaction 2- goes through an intermediate called aconitase

Isomerization of citrate (isocitrate).Reaction 3- isocitrate dehydrogenase

Used to lose hydrogen when oxidizing NADH

Lose a C to CO2 known as carboxylative dehydration and a reduced electron carrier.

Reaction 4- - ketgluterate dehydrogenase complex (very similar to PDH)

Used to lose hydrogen when oxidizing NADH

Lose a C to CO2 known as carboxylative dehydration and a reduced electron carrier.

2 carbons came in as a carboxyl group and 2 carbons left as carbon dioxide. And left with a 4 carbon compound using Coenzyme A.

Very similar to PDH

Made succinyl CoA

Reactions 1-4

Condensation (oxaloacetate and Acetyl CoA)

Isomerization (citrate to isocitrate)

Decarboxylation of isocitrate: CO2, NADH

Decarboxylation of -ketoglutarate: CO2, NADH Reaction 5- Succinyl-CoA synthetase Conversion of succinyl-CoA to succinate

Succinyl CoA is broken down from a 6 carbon chain to a 4 carbon chain with the addition of energy (GTP) and phosphate to produce succinate coenzyme A and GTP wich is then rapidly converted to ATP.Reaction 6- succinate dehydrogenase

Succinate is transferred to an electron carrier FAD which is permanently bound.

All citric acid cylcle enzymes are free to move around EXCEPT fumerase.

Reaction 7- Fumarase Hydration fumarate to malate

Fumerate has to be in the trans position to produce L- malate this is know as stereospecifity and is highly reversible with the G close to 0. L malate will keep using these products to keep the CAC going and is the basis of homeostasis.

Reaction 8- regeneration of oxaloacetate

Has a huge positive G

Yet goes in the forward reaction because oxaloacetate is continuously being used by enzyme 1 to make citrate.

CAC summary

2C acetyl CoA condenses with 4C oxaloacetate to form 6C citrate

Coenzyme A is released

Isomerisation of citrate

Oxidative Decarboxylations (NADH, CO2)

4/8 reactions are oxidations

Energy of oxidation efficiently conserved in reduced carriers: NADH and FADH2

A series of reactions regenerate oxaloacetate

This cycle can be repeated as long as oxygen (for oxidative phosphorylation) and pyruvate are available

8 successive steps

Amphibolic- to be involved in catabolism and anabolism

Different compounds with the CAC can be used to make other compounds such as amino acids, nucleotides, heme groups and gluconeogenesis

Red arrows indicate anaplurotic meaning if these compounds are running low then the CAC cannot turn. Needs regulation- PDH is turned off when there is plenty of energy in the cell which is determined by ATP and NADH.

The Presence of fatty acids means alternate energy is available and glucose doesnt need to used and is stored as glycogen..

CAC is turned on when there is a lot of ADP indicating low level of energy and not much ATP.

Need to have the system making ATP

Lecture 6 oxidative phosphorylation Oxidative phosphorylation- oxidative refers to redox electron carriers and reduction of energy as electrons move through a system, which powers the phosphorylation of ADPATP. Coupling of electron carriers and donating energy, that energy powers the phosphorylation of ADP ATP. EXERGONIC AND ENDERGONIC REACTION, final step hydride ions are used to make water and are oxidized. OILRIG

OIL- OXIDATION IS LOSS

RIG- REDUCTION IS GAIN ALWAYS COUPLED because electrons cannot be free to move around.

Electrons binding with hydrogen to produce hydride ion (2 protons and 1 electron) is a 1 electron carrier. Dehydrogenation- transfer of a hydride ion to NAD NADH

LOTS OF LOST ELECTONS

C-H BONDS RELEASES A LOT ENERGY, THAT ENERGY IS USED TO FUEL THE CELL IN THE ELECTRON TRANSPORT CHAIN!!!

Common electron carriers:

1. NADNADH 2. FADFADH2

As the electron moves down the cascade its losing energy as it reaches water (green oxidized carrier) lowest energy state and is last electron acceptor and most oxidized form. Water has no c-h bonds and is fully oxidized.Electron Transport Chain- in the Matrix of the mitochondria is like the cytoplasm and contains many enzymes. The matrix is where all the pathways of fuel oxidation meet.The ETC is basically taking electrons putting them into the ETC and producing protons across the inner membrane and regenerate NAD to continue the CAC.

Start of ETC- Complex I- NADH dehydrogenase takes NADH NADH2

Complex III aka BC1 complex- contains cytochromes B and C1.Complex IV- contains cytochromes oxidase which oxidizes cytochromes.Complex II- membrane bound proteins- receive NADH and FADH2 which are transported and pumped through this complex. Succinate dehydrogenase from CAC, produces FADH2 which is non mobile and can only be distributed via the ETC. Chemiosmotic gradient aka proton gradient- receives ions from complex 11 and forms the chemiosmotic gradient, powers ATP synthesis.Q and cytochrome C- are mobile electron carriers. As they pass to carriers protons are pumped across the membrane. These electrons which are hydride ions bond with oxygen to produce water.Last step powers the motor for ATP synthesis- rotational catalysis and proton gradient where there is more protons in the inner membrane space than the matrix, they will travel down the concentration gradient, however the membrane is impermeable to ions therefore they have to transfer down a transmembane protein ATP synthase. As they travel down the gradient they power rotational catalysis which leads to ADP ATP.Cytochrome Oxidase- complex IV pumps hydrogen from the matrix to the inner membrane space and takes hydrogen from the matrix to make water. Therefore causing a change in charge in the proton gradient.

Complex I and III are proton pumps THE ETC PRODUCES A PROTON GRADIENT WITHIN THE INNER MEMBRANE OF THE MITOCHONDRIA. THEY DUE THIS DUE THE MEMBRANE BEING IMPERMEABLE TO IONS!!!

1. NADH and FADH2 enter via dehydrogenation NADH donates 2 electrons at complex I (NADH dehydrogenase) and FADH2 donates 2 electrons at complex II (not shown in diagram).2. Coenzyme Q (ubiquinone) lipid soluble and can diffuse through the inner membrane and cytochrome C is mobile and sits at the junction of the inner membrane and inner membrane space and is a one electron carrier are membrane bound carriers electron carriers. Coenzyme Q takes electrons from complex I- Complex III an Complex II Complex III. Cytochrme C complex II(from CAC and succinate dehydrogenase complex)- complex III (bc1 complex)

Complex I contains FMN- flavin mononucleotide which transports the electrons.Chemiosomotic gradient- creates a proton motive force from concentration gradient and a charge separation from the high proton conentraton in the inner membrane.

ETC- 3 proton pumps and take hydrogen to make water, leads to conc grad and charge separation.

Chemiosmotic model

ATP synthesis happens at complex and is known as ATPsynthase. Its also an ATPase and wants to make ADP and Phosphate from ATP, the hydrogen flow and certain affinaity takes it to the right. Made of 2 componants F0 hydrogen pore and F1 Catalytic Head stork joins both componants. Catalytic head is in the matrix and hydrogen pore is in the membrane space. Whole process is controlled by the needs of the cell and is regulated by the ADP and ATP and NAD and NADH ratio so turning on off metabolites. 32 ATP generated and about 65% efficient as some energy is lost as heat.

Lecture 7 Glycogen Metabolism Glycogen main storage of glucose in the liver and some in the skeletal muscles. Comprised of long chains of glucose subunits.

Carbons 1,4 and 6 form the chains on the glucose molecule. Via glycosidic bonds carbon 6 and 1 forms branches in the chain

Livers buffers blood glucose levels by distributing glucose to other tissues were needed whilst muscle glucose is only used in he muscle.

Glycogenolysis- breakdown of gglycogen

Glycogensis- make new glycogen moleculesGlycogen breakdown:

Enzyme 1: Glycogen phosphorylase- main enzyme used to catalyzes the phosphorolytic cleavage of the (14) glycosidic linkage of glycogen releasing a glucose-1-phosphate can only work on single chains not braches. Stops cleaving at 4 molecules before the branching at 1,6 carbon molecule. Enzyme 2: glycogen debranching enzyme- 2 independent acting sites Transferase activity- transfers 3 of the 4 glucose molecule and transfers them to the end of the chain while leaving the other glucose molecule at the branch point.

Glucosidase activity- cleaves last glucose molecule freeing up the rest of the chain to be catalyzed by enzyme 1.Enzyme 3: phophoglucomutase- catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate so it can be used in the production of glucose via glycolysis in skeletal muscle and dephosphorylated in the liver by glucose 6 phosphatase and is not found in the skeletal muscle and released in the blood.

Synthesis of Glycogen (glycogenesis): Prominent in liver and skeletal muscle Reaction 1: via glucose +ATP ADP+ G6p

Reaction in muscle is catalyzed by enzymes hexokinase I and II

Reaction in liver is catalyzed by enzyme Hexokinase IV

G6p is converted to G1p- reversible reaction Synthesis of UDP glucose made from UDP-glucose phosphorlyase enzyme (uric diphosphate) activated sugar nucleotide from G1p and UTP. The UDP glucose is what creates the long polymer chains of glucose to make glycogen. More energetic favorable than glucose. Also used in carbohydrate chains. Glycogen synthase enzyme- catalyzes the transfer of the UDP glucose to the end of the chain to make the chain longer and UDP as it loses the glucose to the chain. Continuously does that to grow the chains and forms (14) glycosidic bonds. Can form 1,6 carbon for branching Branching of glucose chains increases the solubility of the polymer and creates free ends for enzymes to cleave when glucose is needed rapidly.

Add branches by glycogen synthase by moving upto 7 glucose residues to the 1,6 carbon and forms a chain, however cannot start a new polymer.

glycogenin enzyme is the primer that starts a new polymer and also catalyzes the assembly of the polymer. Contains a tyrosine molecule that forms a glycosidic bond with carbon 1 from UDP glucose molecule. This is added to the glycogenin until chain is 6-10 glucose molecules long and glycogen synthase can catalyze the chain.

Regulation of glycogen synthesis and breakdown Coordinately regulated so both mechanism are coordinated at the same time.

Usually regulated when one is more active the other is less active.

Glycogen synthase enzyme- synthesizes glycogen Glycogen phosphorylase enzyme breaks down glycogen

Both enzymes are reciprocally regulated by:1. Allosteric effectors- glycogen phosphorylase can either be in relaxed and active conformations. Molecules such as AMP, ATP and G6p all interact with this enzyme to alter its function: AMP-relaxed conformation and binds when ATP is depleted

ATP and G6p- tense conformation inhibit glycogen phosphorylase

When ATP and G6p are in abundance glycogen breakdown is stopped.

These processes are reversed in glycogen synthase enzyme and are active when G6p and ATP are high.2. Reversible phosphorylation- when phosphate is added to an enzyme can alter catalytic activity. Phosphate is added to 3 types of amino acids, serine, tyrosine and threonine in a protein chain which is donated by ATP. Phosphorylated by a protein kinase and is a reversible reaction. Each kinase can activate and deactivate each enzyme. In glycogen phosphorylase and glycogen synthase can be activated and deactivated by phosphorylation.3. Hormones-

Insulin induces synthesis of glycogen- initiates dephosphorylation cascade of proteins opposite effect of glucagon. Glucagon, epinephrine adrenalin induce breakdown of glycogen.

Hormone regulation 1- ATP is synthesized to cAMP when glucagon binds to adenylate cyclase after a second message is triggered when hormone binds to receptor. This happens all in sinew. Hormone regulation 2- second message binds to PKA (protein kinase A ) which adds inorganic phosphate to proteins and phosphorylates and modifies activity of the protein which basis the physiological activities of glucagon. Introduction to lipids Organic biomolecules with insolubility in water. Derived from fatty acids Major classes:1. Storage lipids- triaclglycerols2. Structural and functional (phospholipids and eicosanoids)

Constitute all cells.

Triglycerides major energy source and storage

Signaling molecules paracrine cells eicosanoids

Lipid based hormones

Fatty acids are components of lipids are long hydrocarbon chains various numbers of double bonds and terminate with a carboxylate group

Phospholipids are made of fatty acids.

Double bonds are usually in cis configuration of fatty acid chains

The more unsaturated the more fluid the chain is.

Numbered fro carboxylate group 1 is the carbon on the carboxyl group and methyl group at the end of the chain is however long the chain is and also named alpha, beta etc carbon

Tryacyl indicates triglycerides stored as fat in adipose cells high level energy source and insulation have 2 tryl groups attached to 3 hydrocarbon chains that can contain 3 R groups on each head on the glycerol molecule.

Sterols

Non fatty acid containing lipids such as cholesterol (major sterol) and maintain structure of membrane.

Signaling molecules in cells eg eicosanoids are paracrine hormones not endocrine and act close to the tissue their made in and derived from fatty acids, involved in clotting, inflammation an immune defense. Derived from archidonic acid that is a 20 carbon chain with 4 double bonds. 3 classes of eicosanoids derived from archandonate:

1. Prostaglandins (prostate gland)- immediating pain and fever and immunosuppresion2. Thromboxanes (platelets)- blood clotting

3. Leukotreines (leukocytes)- 3 conjugated double bonds tri-3 ene- double bond and mediate airway constriction

Archadonate is the second chain triglycerides and is cleaved by the enzyme phospholipase A2 in response to hormonal or other stimuli to synthesize the eicosanoids through a series of reactions.

Formation of prostaglandins and thromboxanes is produced by PGH2 that is synthesized in 2 reactions that are catalyzed by cyclooxengase (COX) 2 ioforms of COX so COX I and COX II, pain relief medication acts on COX. Is both a precursor to prostaglandins and thromboxanes Synthesis of luekotrienes begins with enzyme lipooxygenases found in leukocytes heart, brain lung and spleen

All reactions happen in the phospholipid

COX I- responsible for synthesizing prostaglandins that regulate gastric mucin

COX II- synthesis prostaglandins that mediate pain and fever

Pain medication blocks both COX therefore causing gastric problems such as ulcers

COX is inactivated by aspirin irreversibly until the COX is degraded by the cell and a new COX is made.

COX 2 drugs were specifically targeted so stomach isnt affected for patients with chronic pain have found to increase the chance of strokes and heart attack.

Lecture 8 Fatty Acid Metabolism Fuel molecule part of triglycerides are fatty acids.

Are the HYDROCARBON CHAINS!!

Highly reduced and anhydrous, varying degrees of saturation terminate with a carboxyl group.

Even number of carbon chains most common 16 and 18

Egs Stearic acid 18 carbon chain

Monounsaturated fatty acid single double bond HC chain. Eg (18:1) one double bond in 18C chain.

Cis configuration

Trans configuration made chemically

Attached to a glycerol molecule and stored in adipose tissue, to utilize molecule has to be cleaved ester bond from glycerol group. There is twice the amount of energy in fatty acid than carbohydrates.

Glucose quick energy source Fats long lasting energy source months

Sources of triglycerides:

1. Dietary fat- high fat consumption in food

2. Fat stored in adipose tissue- make fat synthesis and store triglycerides and mobilizing triglycerides when energy is needed

Utilization of Fatty Acids as Fuel

3 stages

1. Mobilization of fatty acid from glycerol ester

2. Activation and transport to mitochondria from the cytosol (for catabolism)

3. Degradation of fatty acids by oxidation to produce ATP for the cell

Mobilization- triacylglycerols are DEGRADED to fatty acids and glycerol by enzyme triacylglycerol lipase. Released from adipose tissue and then transported bound to albumin. Stimulated by hormones epinephrine and glucagon and inhibited by insulin. Glycerol a byproduct of the breakdown of fatty acid is absorbed by the liver and converted to glyceraldehyde-3- phosphate thats used in glycolysis or gluconeogenis. 2. Activation and Transport- activated by being attached to Coenzyme A to form fatty acyl CoA (activated fatty acid that can be transported into the mitochondria), which is catalyzed by enzyme acyl CoA synthase. Carnitine transport the acyl CoA cells in the mitochindria

Any fatty acid that becomes activated it is transported into the mitochondrial matrix for degradation via oxidation.

3. Fatty acid degradation by oxidation- degraded by a recurring sequence of 4 reactions:

1. OXIDATION by FAD- oxidation of fatty acid chain by enzyme Acyl CoA dehydrogenase to give Enoyl CoA. FAD FADH2 reaction is on the BETA CARBON, 2 hydrogens are removed. Trans form molecule2. HYDRATION- add water on double bond formed by oxidation of beta carbon, stereospecific reaction only L isomer is formed3. 2nd OXIDATION by NAD+- removes 2 hydrogens one from water and one from carbon to produce C=O bond.4. THIOLYSIS by Acyl CoA- cleaving off 2 carbon unit in the form of acetyl CoA, making the chain 2 carbons shorter. A 16 carbon chain the 4 reactions will happen 7 times to produce 8 molecules of acetyl CoA, will enter CAC and produce NADH and FADH2 and both electron carriers enter the electron transport chain to produce ATP. And gives 108ATP molecules and comes from FAD and NAD being passed in the ETC to produce the ATP. 8 molecules Acetyl CoA

All move in CAC

Each Acetyl CoA in CAC gives 3 NADH , 1 FADH2 and 1 ATPEven number carbon chains produce total carbon number/2 acetyl CoA eg 16C= 8 acetyl CoA

Odd number carbon chains produce (carbon number/2) -1 end product 2 carbon molecule propionyl- CoA. Eg 15C= 6 Acetyl CoA and 1 priopionyl CoA and formed into succinyl CoA which then goes into CAC.Fatty Acid Synthesis

Occurs in 2 stages:Stage 1- formation of malonyl CoA (3C unit)

Stage 2- Elongation of Chain (2C units at a time)

Stage 1- acetyl CoA is converted to malonyl CoA, which makes the chain were 2C from the malonyl CoA molecule is added, is catalyzed by the enzyme acetyl transacylase, this is an irreversible reaction in fatty acid synthesis . This step is a MAJOR step in regulation.Step 2- Elongation of chain- occurs in recurring series of 4 reactions, this is opposite to - Oxidation reaction, one catalytic enzyme that controls all polypeptide reactions. Long flexible arm of fatty acid chain is attached to ACP, which is attached to the enzyme. Multienzyme complex with 3 enzyme domains.1. Condensation- produce 4C using acyl-malonyl ACP condensing enzyme2. Reduction- - ketoacyl ACP reductase3. Dehydration- removes water4. 2nd Reduction- enoyl ACP reductase enzyme End product is palmitate C16 + ACP chain, modifications to the chain such as extensions and double bonds are done by other enzymes in the cell.

Degradation synthesis

1. Occurs in mitochondrial matrixOccurs in the cytosol

2. intermediates of - oxidation are linked to acetyl CoAIntermediates are attached to ACP

3. separate enzymes catalyze each stepOne multienzyme complex (fatty acid synthase)

4. oxidants NAD+ and FADH usedReductant NADPH used

5. variety of chains can be degraded Stops at 16C palmitate is synthesized

Regulation of Fatty Acid Metabolism Regulated to physiological needs of the cell.

Synthesis is maximal when carbs and energy are plentiful after a big meal.

Acetyl CoA Carboxylase initiates fatty acid synthesis and catalyzes the committed step and regulated by signals, allosteric effectors and covalent modification. ACTIVATED BY DEPHOSPHORYLATION, DEACTIVATED BY PHOSPHORYLATION INSULIN- SYNTHESIS FATTY ACIDS GLUCAGON & EPINEPHRINE- INHIBITS SYTHESIS AND INIATES BREAKDOWN OF FATTY ACIDS.Lecture 9: Amino Acid Metabolism Protein metabolism

2 molecules that have nitrogen-amino acids and nucleotides

Amino acids are incorporated into proteins on ribosomes.

Amino acids all contain a -carbon with amino group and carboxyl group attached to the carbon and a side chain. All have a different side chain.

20 amino acids incorporated into proteins.

3 groups: hydrophobic side chain, hydrophilic side chain and in between side chains

Peptide bond that joins 2 amino acids together formed on the ribosome during protein synthesis.

Thousands of amino acid chain.

Amino end start of the chain (N terminus)

Carboxyl end of the chain (C terminus)

Amino acids that are used for energy come from dietary protein (meat and certain vegetables).

Metabolism- amino acids cant be stored by the body and only used when there is excess amount of amino acids or a lack of other energy sources.

In order to utilize energy from the amino acids the amino group has to be removed from the -carbon and the remaining amino acid molecule is used for energy.

Amino group that is removed is toxic and has to be eliminated quickly as the nitrogen is toxic and this occurs through the urea cycle.

Serve as a nitrogen source for other compounds that are nitrogen based molecules such as heme in Hb, hormones and phospholipids, neurotransmitters and bases of DNA and RNA. Proteolytic enzymes cleave long chain proteins into smaller peptides that are further hydrolysed into individual amino acids. Cleave the end of the amino end.

Aminopeptidases cleave amino end of the amino acid. Work in one direction Carboxypeptidases cleave the carboxyl end of the amino acid work the opposite direction to the aminopeptidase. Endopeptidases hydrolyze specific bonds within a protein chain (trypsin create new ends).Degradation of Amino Acids and Urea Cycle Amino acids are degraded when there is normal protein turnover. body protein is constantly being renewed which leaves excess amino acids. Amino acids are utilized for energy. When carbs are scarce such as starvation or diabetes mellitus.

Amino acids are converted to urea in the liver. In muscle excess amino groups are picked up by pyruvate and converted to alanine, and moved to the liver which converted by into pyruvate for glycolysis and transferred back into the muscle.

Alanine (specific to muscle) and glutamine are carriers on amino acids.

Degradation of amino acids occurs in 2 stages:

1. Removal of NH2 group via aminotransferases enzymes (different enzyme for amino acids have same mechanism) the reaction entails the -amino group is transferred to the -carbon of the -ketoglutarate, which is converted to glutamate and -keto acid. This reaction will happen to any amino of the 20 amino acids. Urea Cycle 5 steps takes free ammonia and converts it to urea - begins in the liver mitochondria, 3 steps in the cytosol, expands 2 cycles mitochondria and cell cytosol!! AS SOON AS GLUTAMATE ENTERS THE MITOCHONDRIA ITS DEAMINATED THE FREE N2 IS CONVERTED TO UREA AND QUICKLY REMOVED BY THE UREA CYCLE 1. Formation of carbamoyl phosphate in mitochondrial matrix ( enzyme carbamoyl phosphate synthase) 2. Formation of citrulline and passes through the mitochondria into the cytosol.3. In the cytosol the second amino group of urea is attached from aspartate, example of an amino acid donating an amine group to another compound to produce another compound, production of argininosuccinate 4. Cleavage of argininosuccinate produce arginine and fumerate, once fumerate is formed can reenter the mitochondria CAC intermediates. FUMERATE JOINS CAC AND UREA CYCLE!!5. Arginine is cleaved to produce urea and ornithine.

Image showing the CAC and Urea cycle overlapping each other with argininosuccinate

2.

Breakdown of carbon skeleton that is left behind when the amino acid is deaminated.

The 6 products that remain when amino acids are broken down are the 6 (1.Actyl CoA,2 -ketoglutarate,3. Succinyl CoA, 4. Fumarate,5. Oxyloacetate and 6. Pyruvate) constituents of the CAC. All products from deamination enter the CAC at different points, the remaining carbon skeletons are oxidized to CO2 and water.

Glucogenic amino acids degraded to -ketoglutarate, succinyl CoA, Fumarate, Oxaloacetate and pyruvate. C skeletons used to produce glucose Ketogenic amino acids- degraded to Acetyl CoA acetylacetate CoA, C skeletons used to produce ketone bodies (produce under starvation).

Regulation of Urea Cycle: Flux of N2 through the cycle varies with diet, more protein more active the urea cycle.

Prolonged starvation, breakdown of muscle protein thusly more nitrogen is produced. Rates of synthesis of enzymes within the urea cycle increase when high amounts of protein are consumed. Some organisms excrete nitrogen as nitric acid. (birds and reptiles)

Biosynthesis of Amino Acids Synthesis requires nitrogen, which is salvaged or reused. The nitrogen cycle Humans rely on plants and microbes to turn nitrogen in a digestible form such as ammonia to produce amino acids. 2 principle enzymes that convert ammonia into amino acids1. Glutamate corresponding enzyme Glutamate dehydrogenase forms glutamine2. Glutamine corresponding enzyme Glutamine synthetase forms glutamine Both can be transaminated to produce amino acids and glutamate can transfer its amide group to other amino acids. Glutamine synthetase is the most important pathway for incorporating ammonia into glutamine. Regulated allosterically and covalent modification. Highly regulated as it synthesizes all amino acids. Essential amino acids are those that CANNOT be synthesized so we get them from diet. Non-essential we can synthesis in the cell. All amino acids are derived from GLYCOLYSIS, PENTOSE PHOSPHATE PATHWAY AND CAC. 6 precursors that amin0 acids are made -ketoglutarate, 3- phosphoglycerate, oxyloacetate, pyruvate, phosphophenolpyruvate & erythrose-4-phosphate and ribose-5-phosphate. Precursor 1- -ketoglutarate Precursor 2- 3- phosphoglycerate Precursor 3- oxyloacetate, Precursor 4- , pyruvate Precursor 5- phosphophenolpyruvate & erythrose-4-phosphate Precursor 6- ribose-5-phosphate

Lecture 10: Nucleotide Metabolism1