Chapter 9

Catabolic Pathways and Production of ATP

-Aerobic respiration consumes organic molecules and O2 to yield ATP -Anaerobic respiration is similar to aerobic respiration but consumes compounds other than O2 (not the same as fermentation) -Cellular respiration includes both aerobic and anaerobic respiration but is often used to refer to aerobic respiration

The Stages of Cellular Respiration: Preview

-Glycolysis takes place in the cytosol, responsible for the conversion of glucose to pyruvate -This processes generates ATP through substrate-level phosphorylation -Under right conditions, pyruvate can be shuttled into the mitochondrial matrix and result in pyruvate oxidation to from Acetyl CoA -In the mitochondrial matrix, Acetyl CoA is used to initiate the citric acid cycle, which generates ATP through substrate-level phosphorylation -A significant outcome of this cycle is the production of reduced electron carriers (NADH and FADH2) which moves into the process of oxidative phosphorylation (along with electrons from glycolysis) -Oxidative Phosphorylation is composed of 2 parts, the electron transport chain and chemiosmosis -Both events work together to produce significantly larger amounts of ATP than through the substrate-level phosphorylation during glycolysis or citric acid cycle -All of the events occur in the mitochondria, but depends on location

Glucose is a good potential source of energy

-The oxidation of glucose is highly exergonic -When it comes to the oxidation of sugar, △G = -686 kcal/mol is based on the complete oxidation in the presence of oxygen -The △G is the same whether or not we go through the process of stepwise oxidation or direct burning -In both scenarios, we have the same level of free energy in the reactants vs the product, so the amount of free energy released is identical -The difference of acquiring the energy is essential -The uncontrolled burning is not compatible with the needs of life since free energy released by this oxidation event is unusable (released as heat)- -Stepwise oxidation allows us to conserve as much usable energy as possible, conserved in the bonds of ATP within the release of discrete quantities

Pyruvate occupies a key position as a branching point between processes

1) Aerobic Cellular Respiration (O2 present) 2) Fermentation (No O2 present) Thus, the fate of pyruvate is dictated by the availability of oxygen -What is produced during fermentation will be dictated by what type of organism that is involved in that process -Production of pyruvate is directly linked to glycolysis; glycolysis and fermentation are separate events -You cannot include glycolysis in the process of fermentation -Fermentative processes depend on the pyruvate produced at the end of glycolysis -The goal of fermentation is to regenerate an oxidized carrier to head back into glycolysis, allowing us to create energy in the form of ATP -Thus, fermentation allows us to continue the process of glycolysis in the absence of oxygen

The Principle of Redox

A redox reaction includes both the oxidation (loss of electrons) and reduction (gain of electrons) events -The electron donor (oxidized) can also be referred to as the reducing agent -The electron acceptor (reduced) can be referred to as the oxidizing agent

Oxidative Phosphorylation (Electron Transport Chain: Electrochemical Gradient)

A voltage gradient has been generated in the inner mitochondrial gradient (membrane potential) -The matrix facing side would be negative, and the inter-membrane space side would be positive due to the unequal distribution of ions across the bilayer (more on the inter-membrane space side) A pH gradient also exists across the membrane -As more protons enter the inter-membrane space, the pH will decrease, and will increase within the matrix due to loss of those protons The summation of both of these forces (unequal distribution of ions and pH gradient) is going to create an electrochemical gradient that will generate a desire for protons to head back down their gradient into the matrix -When it comes to this desire to head back down their concentration gradient, the inner mitochondrial membrane is not as permeable as the outer membrane -Thus, we would not expect these protons to be able to simply diffuse through the phospholipid bilayer

CONCEPT 9.3

After pyruvate is oxidized, the citric acid cycle completes the energy-yielding oxidation of organic molecules

CONCEPT 9.1

Catabolic pathways yield energy by oxidizing organic fuels

The Citric Acid Cycle

Completes the breakdown of glucose by oxidizing a derivative of pyruvate to carbon dioxide. -Takes place within the mitochondrial matrix -An important intermediate within the citric acid pathway is citrate, the ionized form of citric acid -Because citrate has 3 carboxylic acid groups, this process is sometimes referred to as the tri-carboxylic acid cycle (TCA cycle) or the Krebs Cycle -Two carbons enter the cycle from Acetyl CoA, joining with oxaloacetate (a 4-carbon acceptor molecule) to form the 6-carbon compound citrate -Decarboxylation events occur at 2 steps within the cycle so that the two carbons that came in are balanced by two molecules of carbon lost in the form of carbon dioxide -Oxidation occurs at 3 steps within the cycle, -NAD+ serves as an electron acceptor at 3 points in the cycle, producing NADH -FAD serves as an electron acceptor at 1 point, resulting in FADH2 -ATP is generated at 1 point, which will have GTP as an intermediate in animal cells -One one turn of the Krebs Cycle has occurred, there will be a regeneration of oxaloacetate, ready to accept another 2 carbon molecule to start the process again -Because 2 molecules of pyruvate are produced due to them entering after glycolysis. This means that the citric acid cycle occurs twice in order to metabolize the products that result from one glucose molecule -The point of the citric acid cycle is to make a lot of reduced carriers to head on into the electron transport chain to serve a role in production of ATP through oxidative phosphorylation

Oxidative Phosphorylation (Electron Transport Chain: Complex 1)

Complex 1: the transfers from electrons from NADH to Complex 1 -Complex 1 (transmembrane proteins) transfers electrons to coenzyme Q (found in the interior of the mitochondrial inner membrane) -During the transfer of electrons in Complex 1, protons are pumped from the mitochondrial matrix out into the inter-membrane space -Energy released from the exergonic movement of these electrons provides the energy needed to move protons against their concentration gradient

CONCEPT 9.6

Glycolysis and the citric acid cycle connect to many other metabolic pathways

Oxidative Phosphorylation (Electron Transport Chain: Complex 2)

Complex 2: receives electrons from FADH2 and transfers them to coenzyme Q -Complex 2 is embedded in the inner membrane but does not expand through to the outer membrane, making it integral, but not transmembrane -Thus, complex 2 cannot function as a proton pump, and will not have a direct contribution to the electro-chemical gradient used to generate ATP later

Oxidative Phosphorylation (Electron Transport Chain: Complex 3)

Complex 3: accepts electrons from coenzyme Q, then transfers them to cytochrome C -As the electrons pass through Complex 3, protons are pumped into the inter-membrane space -The energy of electron moving from a higher state of free energy to a lower one is what allows for protons to head out into the inter-membrane space against their concentration gradient (moving protons to an area of higher concentration)

Oxidative Phosphorylation (Electron Transport Chain: Complex 4)

Complex 4: receives electrons from cytochrome C and passes them to oxygen -This is a transmembrane protein, so protons will be pumped out into the inter-membrane space here as well Coenzyme Q: a carrier within the electron transport chain that is able to interact with the contents from Complex 1 and 2 -It is the one carrier within this process that is not a protein; it is freely mobile within the nonpolar interior (hydrocarbon tails) of the inner mitochondrial membrane Cytochrome C: a peripheral membrane protein; loosely associated with the outer surface of the inner mitochondrial membrane -It is capable of diffusing rapidly between these protein complexes, which is beneficial in helping to move electrons from one area to the next Complexes 1, 3, and 4 play a role in pumping (active transport) protons against their gradient -Energy conversion that results in passing electrons between these complexes is harness to pump protons across the inner membrane -This gradient is what drives ATP synthesis by ATP Synthase (chemiosmosis) -The movement of electrons in the electron transport chain generates a proton motive force (causes the cell to act as a batter)

An Account of ATP Production by Cellular Respiration

During cellular respiration, most energy flows as glucose, NADH, electron transport chain, proton motive force, ATP synthase -Efficiency of processes can never be 100% -A fraction of the energy found in a glucose molecule can be transferred into these processes and be harnessed to make ATP We make about 30 or 32 molecules of ATP through this process, and the rest of the energy that results from the largely exergonic process is lost as heat -There are certain reactions that are not directly coupled, resulting int he ration of NADH to ATP not being a hole number -ATP yield varies depending on whether electrons are passed to NAD+ or FADH within the mitochondrial matrix -The proton motive force is also used to drive other work

CONCEPT 9.4

During oxidative phosphorylation, chemiosmosis couples electron transport to ATP synthesis

Catabolic pathways release stored energy by breaking down complex molecules

Energy metabolism involves energy yielding oxidative reactions -The oxidation of organic molecules includes the removal of electrons as well as protons -The process of oxidation can be referred to as a dehydrogenation in organic compounds since the removal of an electron and a proton is a hydrogen atom -When there is an oxidation reaction, there will be a reduction reaction -Reduction is the gain of electrons and protons (hydrogenation) -Oxidation and reduction reaction always occur simultaneously

Glycolysis and the citric acid cycle connect to many other metabolic pathways

Fats (large biological molecules) can be broken down into fatty acids and glycerol -In the structure of fatty acids, there will be long hydrocarbon chains, and these bonds have a ton of accessible energy -Fatty acids can be converted into Acetyl groups, which can form Acetyl CoA to feed into the citric acid cycle -This process can be continued until we have broken down all of the different 2-carbon structures that make up a long tail -We can generate far more ATP using fats than with carbohydrates, but carbohydrates are more easily accessible Proteins can be broken down into amino acids, and can enter the process of cellular respiration at different places (glycolysis, acetyl CoA, citric acid cycle) Pathways have several converging intermediates, and link together with the ultimate goal of breaking down organic fuel -Having intermediates in cellular respiration is essential for the inter-conversion of different types of molecules

CONCEPT 9.5

Fermentation and anaerobic respiration enable cells to produce ATP without the use of oxygen

Alcohol Fermentation (Ethanol Fermentation Process)

Glycolysis generates 2 pyruvate molecules -The process of fermentation releases CO2 (decarboxylation event), producing Acetaldehyde -Ej. the release of carbon dioxide is what can cause yeast in bread to rise -Acetaldehyde is capable of accepting electrons from NADH (reduced carrier), generated during glycolysis -Reducing acetaldehyde leads to the formation of ethanol -Ethanol is a byproduct of fermentation; the point of fermentation is tore generate NAD+ to pick up more particles of energy When these byproducts build up they can be harmful: -Excess levels of CO2 in our bodies can cause our blood pH to become too acidic -For organisms like yeast, a buildup of ethanol can become toxic and lead to its death (threshold is ~12%) -Thus, naturally fermented alcohol will usually max out at 12% ethanol due to toxicity of ethanol buildup

CONCEPT 9.2

Glycolysis harvests chemical energy by oxidizing glucose to pyruvate

Stepwise Energy Harvest via NAD+ and the Electron Transport Chain

In biological oxidations, electrons and hydrogens are removed from the substrate and transferred to a coenzyme, which is not consumed in the process and can be recycled -As a result, coenzyme concentrations are relatively low within a cell since they are still capable of meeting cellular needs In dehydrogenation, organic molecules get broken down into a series of steps: NAD+ is one of the essential components that allows for accepting what will be released by oxidation events -NAD+ functions as an oxidizing agents because it is an electron acceptor during cellular respiration -Each NADH that is produces (reduced) represents stored energy to synthesize ATP

Lactic Acid Fermentation

In contrast to alcohol fermentation, lactic acid fermentation does not have a decarboxylation event (CO2 removal) Generation of pyruvate at the end of glycolysis, and animal cells will have an enzyme capable of transferring electrons from NADH to pyruvate -This process will generate lactate (lactic acid), capable of regenerating NAD+ to use within glycolysis Generally, lactic acid being a byproduct of fermentation can be swept away by circulation from the site of production -Lactic acid is produced in muscle cells with dense population of mitochondria that depend on ATP to function -If circulation can't match the rate of lactic acid accumulation, this byproduct can interfere with muscle function, contributing to muscle fatigue -Ej. long distance running where cardiovascular effort requires more oxygen than you can take in, in order to provide enough ATP to drive that work you will divert processes to lactic acid fermentation Situations can exist in which we would lack oxygen and we would try to utilize fermentation but the amount of ATP is insufficient, and would cause death -But, we can have events taking place in our bodies where some cells resort to this process because there is not enough oxygen reaching these cells as opposed to others

Glycolysis: Energy Investment Phase

Input: glucose, 2 ATP Output: pyruvate, 4 ATP (2 net ATP), 2 NADH Step 1) Donation of a terminal phosphate group from ATP to glucose, resulting in glucose-6 phosphate -Hexokinase (catalyzes phosphorylation; implies group specificity) is the enzyme catalyzing the reaction Step 2) A phosphogluco-isomerase modifies glucose-6 phosphate into fructose-6 phosphate -Benefits by adding an additional phosphorylation -Original glucose-6 phosphate is not readily phosphorylated, so the first few reactions of glycolysis the goal is to converted this unphosphorylated glucose into a bi-phosphorylated one

ATP Synthase is a molecular rotary motor

It has a membrane-bound portion and a narrow stock that connects this membrane-bound portion to the catalytic portion (catalytic knob) -The area in the membrane has a channel that allows for the movement of protons down their concentration gradient -The movement of protons into the membrane's complex is going to provide the mechanical energy that is going to drive the catalytic knob, resulting in ATP synthesis -The flow of protons through a channel of the TP synthase can be compared to the hydroelectric example in Chapter 8. Both movements provide the energy that results in rotation In oxidative phosphorylation, we are not depending on an organic substrate in order to directly phosphorylate ADP into ATP -We utilize the available ADP and an inorganic phosphate found within the mitochondrial matrix that we can utilize in order to achieve ATP production Key distinction from substrate-level phosphorylation is that we are harnessing the power of an electrochemical gradient to drive oxidative phosphorylation, we are able to generate far more ATP

Glycolysis results in ATP generation without the direct involvement of oxygen through the catabolism of glucose to pyruvate

It is a 10-step reaction sequence during which we partially oxidize glucose and conserve some of that usable energy that is released to ATP, as well as the reduced coenzyme NADH -Glycolysis = a sweet splitting Glycolysis occurs within the cytosol, where we initiate the process of glucose oxidation, but not where it is completed

Electron Transport Chain

Located in the inner membrane of the mitochondrion -There are many components involved in this process, many of which are proteins that receive electrons from NADH and FADH2, alternating between a reduced state and an oxidized state -There is a shift with respect to free energy, higher state to lower state -This drop will be from the source of organic nutrients to oxygen in small steps to release energy in manageable amounts -NADH will be okay removing these electrons, and with each progressive step forward, each component wants that electron more than the ones that came before it -The most electronegative component in this process will be oxygen; it will serve as a really strong oxidizing agent which will result in the production of water -Although water is not the point of this process, it is generated through the behavior of oxygen -Electron carriers function in a sequence determined by their reduction potential (ability to strip electrons away from one another); how electronegative these factors are

Oxidation of Pyruvate to Acetyl CoA

Pyruvate will travel through a porin found in the outer membrane of the mitochondria (it is pre-permeable) -It will then go through a proton-pyruvate symporter found on the inner membrane allowing it to reach the mitochondrial matrix Multi-Enzyme Complex (Pyruvate Dehydrogenase): allows us to organize a series of enzymatic steps so that any chemical intermediates generated won't diffuse away, and subunits can pass from one substrate to the next without releasing chemical intermediates Step 1) carboxyl group removed as carbon dioxide Step 2) remaining carbon fragment is oxidized, and two protons are given away, one to NAD+ (to make NADH) and another a free H+ (aka proton; activated carrier) Step 3) Coenzyme A, a vitamin b derivative, is attached to acetate via its sulfur to make Acetyl CoA

Glycolysis: Energy Investment Phase

Step 3) Fructose 6-phosphate is acted upon by kinase PFK. The kinase phosphorylates with the investment of another molecule of ATP, providing the phosphate group that results in the formation of fructose 1,6-bisphosphate -This reaction is highly exergonic and irreversible -Considered a commitment step in the pathway of glycolysis Step 4) Fructose 1,6-bisphosphate is relatively unstable in this form and would gladly move forward, so an aldolase enzyme catalyzes its cleavage, resulting in two 3-carbon monophosphate sugars Step 5) these trioses are easily converted from one form to another utilizing isomerase; G3P is on a direct path in glycolysis while DHAP is not, so DHAP must be converted to G3P to optimize the oxidation of glucose -The event of aldolase catalyzing fructose 1,6-biphosphate is dictated by the enzyme PFK -PFK's action is considered the rate limiting step in glycolysis (enzymatic regulation, feedback regulation); PFK has several regulatory binding sites which allows it to respond to changes in availability of certain molecules within the cell

Glycolysis: Energy Payoff Phase

Step 6) G3P dehydrogenase will continue oxidizing the molecule, releasing energy to reduce coenzyme NAD+ to NADH Step 7) the one bisphosphoglycerate produced is doubly phosphorylated, and will serve as a substrate that will transfer those substrate groups to molecules of ADP, then forming ATP (1st instance of substrate level phosphorylation) -This generates a 3-phosphoglycerate (a carboxylic acid)

Glycolysis: Energy Payoff Phase

Step 8) 3-phosphoglycerate goes through rearrangement through enzyme phospho-glyceromutase in which position of the phosphate changes; results in 2-phosphoglycerate Step 9) 2-phosphoglycerate goes through dehydration through enzyme enolase (loss of 2 H2O), introducing a double bond; results in PEP Step 10) PEP yields 2 more molecules of ATP through pyruvate kinase; results in 2 molecules of pyruvate (3-carbon)

Substrate Level Phosphorylation

The enzyme-catalyzed formation of ATP by direct transfer of a phosphate group to ADP from an intermediate substrate in catabolism.

There is an initial input of energy in order to get glycolysis going

The initial energy investment provides a payoff that will be worth it through the energy releasing reaction that come afterwards -2 ATP molecules invested, yielding 4 molecules of ATP, 2 molecules of NADH, and pyruvate (potential source of energy) -The only value reduced will be the 2 ATP molecules invested at the beginning, thus a net of 2ATP (end 4ATP - 2 initial ATP = 2ATP net0

Substrate Level Phosphorylation in the Citric Acid Cycle

The linkage between the 4-carbon sixynal group and CoA is a high energy bond -In a coupled reaction, the bond is cleaved and the energy released drives the phosphorylation of guanisine-diphosphate (GDP), resulting in guanisine-triphosphate (GTP) -GTP can transfer a phosphate to ADP, forming ATP -GTP formation is an intermediate; it is energetically equivalent to ATP with respect to their terminal phosphoanhydride bonds, having identical free energy with hydrolysis

The Mitochondria

The outer membrane of the mitochondria is not a significant permeability barrier for ions and small molecules because it contains transmembrane channel proteins (porins), allowing for movement of certain solutes based on size threshold -As a result, the inter-membrane space (gap between outer and inner membrane) will be continuous with the cytosol with respect to small solutes -The pH and environmental conditions will be very comparable to one another -In contrast, the inner mitochondrial membrane does provide a permeability barrier to most solutes -This makes environmental condition in the mitochondrial matrix different from that of the inter-membrane space -Provides discrete compartmentalization within the organelle, but there are certain regions where the inner and outer membranes are in contact with one another, allowing for the movement of certain molecules through the membranes -The formation of cristae (gives more surface area) accommodates large amounts of protein complexes, essential for successful oxidative phosphorylation

Control of Cellular Respiration

The rate of conversion of glucose into pyruvate is tightly regulated in order to meet the needs of the cell PFK is associated with an irreversible reaction and is the first irreversible reaction that is unique to the glycolytic pathway, a commitment towards a pathway that will yield 2 molecules of pyruvate) -This makes PFK one of the most important control elements within the metabolic pathway associated with cellular respiration -Because it is important in regulating whether or not we'll make ATP downstream, it is important to regulate this enzyme -PFK can become more or less active in response to reversible binding of allosteric effectors -They type of regulation will depend on the cell's needs ATP is capable of binding to are regulatory site distinct from the catalytic site of PFK, lowering the affinity of PFK for fructose 6-phsophate, slowing down/inhibiting these processes, preventing us from making an unnecessary supply of ATP through cellular respiration -Harnessing ATP for cellular activities hydrolyzes terminal phosphate group to increase concentration of AMP -Enough concentration of AMP stimulates PFK (allosteric bonding) to synthesize ATP Citrate is produced to initiate the citric acid cycle -The presence of a lot of citric serves as an indication that there is potential to make a lot of ATP (allosterically regulating PFK) ATP and Citrate can serve as negative feedback mechanisms they regulate the enzyme PFK further upstream AMP can serve as a stimulation (positive allosteric regulation), but not feedback because it is not a product of cellular respiration

At this point we have generated a ton of reduced carriers in the form of NADH and FADH2

This accounts for most of the energy that is extracted from the organic molecule -Only modest amounts of ATP are produced during glycolysis and the citric acid cycle through substrate level phosphorylation -ATP synthesis is powered through oxidative phosphorylation

The glycolytic pathway is something that virtually all cells have the ability to do

This extracts energy from glucose by oxidizing it into pyruvate -Under aerobic conditions, pyruvate enters a mitochondrion matrix where oxidation continues -Works under the assumption that oxygen is available in order to drive the transition from the cytosol into the mitochondria