BSCI105 Exam 2 Study Guide
BSCI105 Exam 2 Study Guide Bsci105
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Lecture 11- Enzymes, equilibrium constants and free energy A. Basic cellular metabolism 1000s of biochemical reactions occur each sec in every cell Molecules being synthesized anabolism or biosynthesis (requires energy) Molecules being broken down (degraded) catabolism (releases energy) Synthesizing molecules (forming covalent bonds) requires energy Breaking covalent bonds releases energy that can be used for synthesis ***Key concept is how energy from breaking covalent bonds (potential energy) is used to synthesize covalent bonds. This type of potential energy is called "chemical energy" B. Energy First law of thermodynamics: The total energy in the universe (a closed system) doesn't change can't be created or destroyed. Also, forms of energy are interconvertable (energy from the sun is used to create covalent bonds in glucose in plants) Second law of thermodynamics: entropy in the universe always increases Not all energy released in a reaction is usable. Some dissipates into the surroundings as heat. This energy is disordered (random and unavailable for work). This disorder is called entropy (why there is no perpetual motion machine). Energy is measured in kilocalories (amount of heat energy required to raise the temperature of 1000 grams of water by one degree Celsius) Will talk later about the amount of energy (kilocalories) a degradation reaction releases or a synthesis reaction requires and how coupling reactions that require energy with reactions that release energy is how life works D. Chemical equilibrium OO O All chemical reactions are reversible (in principle) O A OB O P is the reactant B is the product Fructose 6 phosphateO (F6P) O P Glucose 6 phosphate Isomeriz (G6P) Reaction "A" "B" For any chemical reaction, the reaction will proceed in which ever direction favors the formation of the most stable constituents. If B is more stable than A, then at equilibrium, more B will be present than A Equilibrium is a state when no further net change in the concentration of the 76 reactants and products For any reactants and products, the ratio of the two at equilibrium is a constant For any reaction A B Keq = [B] [the concentration of B] Keq is the equilibrium constant [A] [the concentration of A] for the reaction A B The greater (higher) the equilibrium constant, the more the products are favored to form over the reverse reaction of forming the reactants if Keq = 10 then at equilibrium, [B] will be 10 times the [A] if Keq = 1 then at equilibrium, the concentrations of B and A will be the same if Keq = 0.1 then at equilibrium, [B] will be 1/10 the [A] It doesn't matter how much A and B you start with. At equilibrium the ratio of B/A will always be the same (that's what a constant means) If Keq > 1, (the products are more stable than the reactants) the reaction is said to be spontaneous. It does not requi+e energy to proceed. In fact, it releases energy O OH O O Most chemical rePhosphaucose more than one product or reactant. For examOe: O O O P + P Glucose 6 phosphate (G6P) Keq = [glucose] [phosphate] Keq = 260 [glucose 6phosphate] [H 0] 2 The normal stable form of a molecule (also called the "ground state") has a certain amount of free energy (G). In a chemical reaction, the molecule is changed into one or more other molecules that also have a characteristic amount of free energy. The change (difference) in free energy between the initial molecule(s) and the final molecule(s) is a value known as G. You can measure the amount of energy released in a chemical reaction by measuring the difference in energy between the reactants and products. 77 The amount of energy released is related to the Keq The relationship between Keq and G is as follows: G = RT ln Keq Where R is the gas constant (2.0 kcal/mole) and T is the o temperature (298 K). So, G = 1.36 log Keq If Keq is 100, the G is 2.72 kcals/mole Reactions that release energy are called exergonic G is negative, reaction is spontaneous, Keq is >1 Reactions that require energy are called endergonic G is positive, reaction is not spontaneous, Keq is <1 E. Reaction rates, or, what do enzymes do? The reaction rate is the speed at which the reaction proceeds towards equilibrium Even the most favorable (high Keq) reactions, where the products are much more stable than the reactions, never just occur in nature. They need some input energy (activation energy) to get going (get past the energy barrier). Chemists do this by heating reactants (heat is input energy). ***In biology, special proteins called enzymes lower the energy of activation of reactions so that they can occur (lower the activation energy) How enzymes lower the activation energy: Chemical reactions involve breaking and forming covalent bonds Enzymes contort the molecule causing the bond to be strained. It "wants" to break to relieve the strain. If the bond is not strained, it will not spontaneously break no matter how much energy is released. In reactions with more than one reactant, enzymes bring the reactants together and put them in the right orientation to form the products. This greatly speeds the reaction. Enzymes do not affect the Keq! They do not affect the G of the reaction! They only speed up the reaction! 78 Lecture 12- Enzyme kinetics and coupled reactions A. Enzymes Enzyme catalyze reactions (make reactions proceed faster by lowering the energy of activation). Enzymes are called biological catalysts Remember, reactions will take place in the absence of the enzyme, eventually (after, a really really long time) The reactants of an enzyme are called substrates The products of an enzymecatalyzed reaction are called products The location of where the substrate binds and the products form in the enzyme is called the active site B. How enzymes function Remember, enzymes are just proteins that catalyze biochemical reactions As discussed in the first half of the course, all recognition (interaction) between molecules (proteins and nucleic acids, protein enzymes and any substrate such as sugars, nucleotides, etc.) involves the same basic principles: hydrogen bonds, hydrophobic interactions, spatial "fitting together", charge attraction and repulsion. Like all proteins, enzymes have a distinctive 3D shape (tertiary or quaternary structure) that is determined by the order of their amino acids. The substrate(s) "fit" into the active site of the enzyme. This causes either (1) the enzyme to change shape stressing the bond to be broken or (2) changes the shape of the substrate causing the bond to be broken or (3) brings two substrates together for bond formation After a biochemical reaction is complete, the product(s) are released from the active site. The enzyme returns to its original shape and is ready to catalyze another one of the same reactions. Enzymes are not altered or destroyed during the reaction 79 n t e f a 0 20 40 60 80 100 R Temperature (ºC) Optimal pH for pepsin Optimal pH (stomach enzyme) for trypsin (intestinal enzyme) (a) Optimal temperature for two enzymes 0 1 2 3 4 5 6 7 8 9 10 pH (b) Optimal pH for two enzymes Optimal temperature for typical human enzyme C. Optimal temperature for enzyme of thermophilic (heattolerant) bacteria 80 o a f t R Enzyme function is strongly affected by physical conditions Temperature and pH can affect the structure of the protein which in turn can enhance or reduce its ability to catalyze chemical reactions Bacteria that live inside you have enzymes that function best at 37 C (98.6 F) o o Bacteria that live near thermal vents have enzymes that function best at very hot temperatures > 70 C. o pH affects whether R groups with carboxylic acids or amino groups are charged. Adding or reducing charges can significantly affect protein structure. D. Enzymes often have cofactors (also called prosthetic groups) associated with their protein portions Cofactors are substances that associate directly with the enzyme and are required for the reactions to proceed Two types of cofactors: 1) Metal ions (e.g., Zn , Fe )+ ++ They can assist the enzyme by adding or removing electrons from the substrate 2) Coenzymes Coenzymes are not proteins! They are small organic molecules that attach to enzymes near or in the active site. Some coenzymes work by helping to form the correct active site. Others change the structure of the substrate to make the substrate more reactive An enzyme that requires a metal ion or coenzyme will not function without the cofactor. Some molecules are cofactors for many different enzymes E. Enzyme kinetics The rate (speed) of an enzyme catalyzed reaction increases with an increasing concentration of the substrate (the more substrate, the more the enzyme will convert it into products) The rate of the reaction increases very fast but then reaches a plateau when the active sites of the enzyme becomes saturated with substrate. At that point, it doesn't matter how much more substrate you add, since there are only so many enzymes The study of the rates of enzymecatalyzed reactions is called enzyme kinetics Maximum rate of this reaction At the maximum reaction rate, all enzymes are tied up with substrate molecules. At this point, adding more substrate does not make the reaction go any faster Rate of enzyme catalyzed reaction 81 Concentration of substrate If the enzyme has a low affinity for the substrates (doesn’t bind the substrates well), the curve shifts to the right. High affinity, the curse shifts to the left. F. Controlling enzyme reactions: Not all biochemical reactions need to occur all of the time For example, if a cell has an excess of the amino acid histidine used for protein synthesis, it doesn't need to synthesize more histidine from precursors at this time To keep the cell from synthesizing histidine when it doesn't need any, at least one enzyme in the biochemical pathways leading to synthesis of histidine must be inactivated [All complex molecules that your cells can synthesize are made by a series of enzyme catalyzed steps known as a "pathway", where the product of one reaction becomes the substrate for the next reaction] The nine steps required to synthesize histidine require nine different enzymes. Inactivating any one of these enzyme in the pathway will stop histidine from being made (enzymes in a pathway are usually shown above the reaction that they catalyze. So enzyme "1" catalyzes the conversion of "A" to "B" 1 2 3 4 5 6 7 8 9 A B C D E F G H I Histidine energy energy energy energy energy Biosynthetic pathways (where complex molecules like amino acids, nucleotides, sugars or fatty acids are made from smaller, simpler precursors) require an input of energy at many steps (covalent bonds are being made; that can frequently require energy). Which of the nine enzymes does it make the most sense to inactivate? Usually only one enzyme in a pathway can be activated or inactivated. This enzyme is called the allosteric enzyme. The allosteric enzyme has at least one site (other than its active site) where controlling molecules bind the enzyme and turn the enzyme on or off. This is called the allosteric site (see below) The first enzyme in a biochemical pathway (either a biosynthetic pathway or a degradation pathway) that commits a series of further reactions to occur in the pathway is usually the enzyme that can be activated and inactivated (turned on and off). Of all the enzymes in a cell, only a small subset are allosteric enzymes and can be activated and inactivated 82 G. How are enzymes activated and inactivated? There are many ways that enzymes are controlled 1) Allosteric control Besides the active site, allosteric enzymes have additional important site(s) called allosteric sites: is a site where controlling molecules (called effectors) bind. The allosteric site does not need to be near the active site Binding of an effector to the allosteric site changes the conformation of the enzyme Changing the conformation of the enzyme caused by the binding of the effector to the allosteric site leads to one of two things: a) An allosteric activator stabilizes the active form of the enzyme. The enzyme can bind its substrate b) An allosteric repressor stabilizes the inactive form of the enzyme. The enzyme can no longer bind its substrate An example of an effector In the biochemical pathway used as an example on the last page, too much histidine was building up in a cell. How does the pathway sense that there is too much histidine? The product histidine is the effector for the first enzyme in its pathway! When the concentration of histidine is too high, it binds to the allosteric site of the first enzyme in the pathway. The enzyme is inactivated and no longer functions. The pathway is shut down (in that cell) and no more histidine is made. This is an example of negative feedback inhibition Allosteric interactions are reversible! Reversible reactions mean that histidine is being bound and released, bound and released, bound and released etc.. If the histidine concentration in the cell drops, then when histidine is released from the allosteric site, it is used up in protein synthesis and there is no longer sufficient "free" histidine to bind to the allosteric site. The enzyme becomes active because no histidine is bound. The pathway begins to function again to make more histidine Nearly every biochemical pathway in every cell is under some kind of allosteric control (but remember, there is usually just one allosteric enzyme in a pathway) 83 Initial substrate (threonine) Active site available Threonine in active site Enzyme 1 (threonine Isoleucine deaminase) used up by cell Intermediate A Feedback inhibition Enzyme 2 Active site of enzyme 1 no longer binds Intermediate B threonine; Enzyme 3 pathway is switched off. Intermediate C Isoleucine binds to Enzyme 4 allosteric site Intermediate D Enzyme 5 End product (isoleucine) Other ways that enzymes are activated or inactivated: 2) Enzymes can be activated/inactivated by covalent bond attachment of a small charged molecule such as a phosphate group Addition of a phosphate group (phosphorylation) with its two negative charges to an enzyme will change the enzymes conformation, either activating it (for some enzymes) or inactivating it (for other enzymes). This is very similar to what happens when an effector binds to an allosteric site (you should know the difference between "binding" a molecule and forming a covalent attachment to a molecule) Phosphate groups are added to enzymes by other enzymes (enzymes, like other proteins, are often substrates of enzymes) Phosphates can be removed from the enzyme by another enzyme. So addition/removal of phosphates is reversible! The examples above (allosteric control, phosphorylation) are natural, reversible ways that enzymes are activated or inactivated in a healthy organism's cells. 84 Manmade chemicals and chemicals produced by pathogens can be deleterious molecules that inactivate enzymes. These agents are called enzyme inhibitors H. Enzyme inhibitors Inhibitors are artificial or foreign "poisons" that disable particular enzymes that just happen to be able to bind to them There are two types of enzyme inhibitors: 1. Competitive inhibitors resemble an enzymes substrate and compete with the natural substrate for binding to the active site of the enzyme 2. Noncompetitive inhibitors do not compete with substrate binding but bind the enzyme somewhere other than the active site. This binding then changes the structure of the enzyme (and the active site) so that the substrate cannot bind Competitive and noncompetitive inhibitors can be either reversible or irreversible 1. Irreversible inhibitors bind to the enzyme so tightly that the enzyme is ruined forever. It will never function again 2. Reversible inhibitors are molecules that bind more loosely to enzymes and can be released (folic acid) Lecture 13- Introduction to metabolism and the importance of ATP A. Metabolism (some of this will review what we have already covered) Metabolism is the sum of the 1000s of biochemical reactions going on in your cells As we have seen, synthesis and degradation of molecules involve a series of enzyme catalyzed steps in what are called biochemical pathways Making a complex molecule from simple precursors is called biosynthesis or anabolism Breaking down a complex molecule to simple components is called degradation or catabolism Making complex molecules (forming covalent bonds) requires energy Degrading complex molecules (breaking covalent bonds) releases energy The energy released by degrading complex molecules is the energy used to biosynthesize other complex molecules! 85 B. Some other important facts about metabolism There are certain central pathways in metabolism that are nearly identical in every organism, from the simplest bacteria to plants to us. Some of these pathways we will cover in this class glycolysis and the citric acid cycle are examples of central pathways This means that these pathways were established before the evolution of eukaryotic cells! Some biochemical pathways are only found in certain organisms For example, most bacteria and green plants can synthesize all 20 amino acids. Synthesis of each amino acid requires a separate biochemical pathway (but some amino acids are precursors for others) Adult humans can only synthesize 11 amino acids. Within a multicellular organism like us, the central pathways operate in every cell However, other pathways may be found in one cell type, or one organ, and not in others C. Energy for biochemical reactions How is energy released from breaking covalent bonds (degradation reactions) used to form covalent bonds (biosynthesis reactions)? The central source of energy cells use is ATP (adenosine triphosphate, the same ATP used in RNA biosynthesis) For the reaction: ATP ADP + Pi G = 7.3 kcal/mol This reaction is exergonic. 7.3 kcal/mol of energy is released and is available to run an unfavorable reaction. Exergonic chemical reactions are called spontaneous. ***However, exergonic reactions still need an enzyme to lower the energy of activation! To make ATP from ADP and Pi, ADP + Pi ATP G = 7.3 kcal/mol Biochemical reactions that require an input of energy are called endergonic If G is a positive number, the reaction is endergonic. If G is a negative number, the reaction is exergonic ATP is used not only for biosynthesis reactions in metabolism, but also for muscle contraction and to transport molecules across membranes (remember active transport?) D. Where did the original energy come from to make the carbohydrates in the food we eat? 86 All energy originates with the sun Plants (and certain bacteria) are able to use the sun's energy to biosynthesize sugars E. How the cell uses ATP to drive nonspontaneous reactions (endergonic reactions) Here is a more detailed explanation of the above figure: 87 88 F. How a cell reforms ATP (from ADP and Pi) Plants have three ways of making ATP, one of which requires oxygen. People have two ways of making ATP, one of which requires oxygen The energy released when certain covalent bonds are broken in the degradation of glucose (i.e., food) can be coupled to the remaking of ATP from ADP and Pi. This is called substratelevel phosphorylation If you are an anaerobic organism (an organism that cannot use oxygen [0 ]) 2r an organism growing under anaerobic conditions (like yeast that you deprive of oxygen or your muscles when you are “out of breath”) then this is the only way you have of making ATP Substratelevel phosphorylation The reaction being catalyzed by the enzyme is: PEP Pyruvate + Pi G = 14.8 kcals/mole ADP + Pi ATP G = 7.3 kcals/mole G for the complete reaction is: G = 7.5 kcals/mole This is the last step of glycolysis However, if you can use O , 2hen you have another way to produce ATP from ADP and Pi This second way (which unfortunately is quite complicated to understand) is called oxidative phosphorylation and involves the transfer of electrons in a series of oxidationreduction reactions G. Oxidationreduction reactions The transfer of electrons (usually in the form of hydrogen atoms) between two molecules or atoms is an oxidationreduction reaction Oxidation and reduction reactions always occur together 89 One molecule (or atom) gains an electron and is said to be reduced The other molecule (or atom) loses the electron and is said to be oxidized The transfer of hydrogen atoms (not ions!) between two molecules is an oxidation reduction reaction because this transfer involves the hydrogen's electron One molecule gains the hydrogen(s) and is reduced One molecule transfers the hydrogens and is oxidized Only a few different molecules in biology can accept the donation of a hydrogen atom (get reduced). The main compound involved in oxidation/reduction reactions that accepts + hydrogen during respiration is NAD (nicotinamide adenine dinucleotide) dehydrogenase Dehydenzymese enzyme N+ Electrons get transferred to NAD to form NADH. The transfer of those electrons from NADH to O re2eases 52.5 kcals of energy! NAD +2H NADH + H + NADH + H + ½ 0 N2D + H 0+ 2 G = 52.4 kcals/mol of energy (52.4 kcals/mol of energy is released) This release of energy is enough to make how many ATP? (Remember that to make ATP from ADP and Pi requires 7.3 kcals/mol of energy) 90 This formation of ATP is called oxidative phosphorylation because it uses O2 as an electron acceptor. Anaerobic organisms (or cells/tissues under anaerobic conditions) cannot use oxidative phosphorylation because there is no O2. The transfer of electrons to O2 requires a series of oxidation reduction reactions that "pass" the electrons through and eventually to O2. Lecture 14- Glycolysis A. Glycolysis Glycolysis is the central biochemical pathway in metabolism Glycolysis is the series of enzymecatalyzed biochemical reactions that lead to the degradation of six carbon sugars such as glucose to the three carbon compound pyruvate When glucose is broken down into two molecules of pyruvate, energy is released that is used to form ATP from ADP + Pi This pathway is essentially identical in all organisms and cells, both aerobic and anaerobic Although we will begin the pathway with glucose, there are enzymes that convert other carbohydrates into glucose or into fructose 6phosphate, one of the early intermediates in the pathway (this will become more clear below) B. Preview of glycolysis The breakdown of one molecule of glucose to two molecules of pyruvate takes 10 biochemical reactions, each one catalyzed by a different enzyme (so 10 enzymes are required) All of the enzymes are found in the cytosol, so glycolysis takes place in the cytosol The breakdown of one molecule of glucose to two molecules of pyruvate produces two n molecules of ATP and reduces two molecules of NAD to NADH 91 C. The steps of glycolysis D. Summary of the main points of glycolysis 1. The first five reactions are endergonic. 2. In two of the first five reactions, ATP is broken down to ADP and the remaining Pi transferred to the sugar. So two molecules of ATP are used up in the first five reactions 3. The two phosphate groups transferred to the sugar in the first 5 reactions will eventually be transferred back to ADP to form ATP 4. In the fourth reaction, the sixcarbon sugar is cleaved into two threecarbon sugars 5. In the fifth reaction, one of the two threecarbon sugars is transformed into the other. At this point, both three carbon sugars are identical and both go through the remaining reactions 6. In the sixth reaction, one more phosphate is added to each of the two threecarbon sugars. Now each sugar has two phosphates 7. The sixth reaction is also a+ oxidation/reduction reaction. Each +hree carbon sugar gets oxidized and 2 NAD are reduced to 2 NADH (one NAD to NADH for each threecarbon sugar) 8. In the seventh and tenth reactions, 2 ADP get converted into two ATP for each of the threecarbon compounds. The 2 Pi that get transferred to ADP to form ATP come from the Pi on the threecarbon compounds. So four ATP are produced. 9. The net number of ATP produced when converting glucose to pyruvate is two The complete reaction of glycolysis under standard conditions (adding up all of the individual steps [the same constituent on both sides of an equation are cancelled out]) is: + + Glucose + 2 ADP + 2 Pi + 2 NAD 2 Pyruvate + 2 ATP + 2 NADH + 2 H G = 17.4 kcals/mol 92 The total G for a pathway is the sum of the G of the individual steps This high negative G means that this pathway is exergonic (energy is released), it occurs spontaneously and is very favorable Acetyl D. How Glycolysis is regulated The enzyme in glycolysis under the tightest regulation is the enzyme in the third step: Fructose 6P + ATP Fructose 1,6 bisphosphate + ADP Phosphofructokinase 93 Why this enzyme? It is an early enzyme in glycolysis (but not the first) Fructose 6P is a major branch point in metabolism 1) Many other sugars feed into the pathway at this point 2) Also, fructose 6P can go through glycolysis, or 3) Fructose 6P can go through a pathway called the pentose phosphate shunt (this is the pathway that makes ribose 5' P, which is found in nucleotides) or 4) Fructose 6P can get converted back into glucose by the gluconeogenesis pathway (see below) ATP represses the enzyme; AMP activates the enzyme E. What about the reverse pathway? The reverse pathway, making glucose from two molecules of pyruvate must therefore require an input of energy in order to proceed. The input of energy comes from ATP and GTP (GTP, like ATP, also contains high energy bonds that can be used to drive unfavorable reactions). F. Overall reaction of the reverse pathway The pathway of making glucose from pyruvate is called gluconeogenesis The overall equation is: 2 Pyruvate + 2 NADH + 4 ATP + 2 GTP Glucose + 2 NAD + 4 ADP + 2 GDP + 6 Pi G = 9.0 kcal/mol Gluconeogenesis is also exergonic overall, but only because you are using ATP (and transfer of H from NADH) for energy! Degradation reactions release energy (that can be used to make ATP from ADP and Pi) Biosynthesis reactions require an input of energy In gluconeogenesis, 10 steps are required: 94 7 steps use the exact same enzymes as glycolysis, but they catalyze the reverse reaction 3 steps use unique enzymes Lecture 15- Citric acid (Krebs) cycle A. The fate of pyruvate Pyruvate can have several different fates depending on the energy level (amount of ATP) in the cell, and whether the cell can use oxygen or not Also, besides making pyruvate from glucose, pyruvate is also produced during the degradation of several amino acids. The fate of pyruvate is one of the major crossroads in metabolism 1) If the cell requires energy (is low on ATP [a low ATP/ADP ratio]), then pyruvate goes into the citric acid cycle (the next pathway that we will cover) so that more ATP can be made 2) If the cell has plenty of energy (has lots of ATP [a high ATP/ADP ratio]), then pyruvate gets converted into glucose by gluconeogenesis, and glucose gets converted into glycogen for storage 3) If the cell cannot use oxygen (e.g., anaerobic bacteria), then fermentation takes place 4) If a cell isn't getting enough oxygen B. The fate of pyruvate during aerobic conditions (oxygen is present and the cell needs energy) 1) First, pyruvate (threecarbons) gets converted into the twocarbon acetate (the third carbon is released as CO ),2and acetate gets linked to a carrier molecule called CoA to form acetyl CoA ***This is an irreversible reaction!! Once pyruvate is converted into acetyl CoA, it can never be made back into glucose! This reaction (catalyzed by multiple enzymes called the pyruvate dehydrogenase complex) is under very tight cellular control. It is only active when the cell needs energy The conversion of pyruvate into acetate is an oxidation/reduction reaction Pyruvate is oxidized to acetate (and CO is released) + 2 NAD is reduced to NADH 95 Gluconeogenesis Fatty acid biosyntTo citric acid cycle Glycolysis C. Pyruvate dehydrogenase is one of the most highly controlled enzymes in the cell. Your brain requires glucose for ATP production. If no glucose is available the brain starves. Cells must regulate when to convert pyruvate into acetyl CoA (irreversible) and when to take the pyruvate back to glucose for storage in the liver and muscles as glycogen. Acetyl CoA Pyruvate Glucose The cell should inactivate pyruvate dehydrogenase if: 1) The cell has plenty of ATP (high ATP/ADP ratio) Activates Inhibits 2) The concentration of acetyl CoA is high (why make more if it's not being used up?) 3) The concentration of NADH is high (why make more if it's not being used up?)MP ATP NAD+ NADH CoA Acetyl CoA 96 Pyruvate dehydrogenase AMP, ATP, NAD, NADH, Acetyl CoA and CoA are all allosteric effectors of pyruvate (as fat) when energy is not needed dehydrogenase (remember, allosteric effectors are molecules that bind to the allosteric site of an enzyme and either activate or inactivate it) AMP, CoA, and NAD are allosteric activators. They activate pyruvate dehydrogenase. So when concentrations of these are high, the cell must need energy (ATP made) and pyruvate dehydrogenase is active! ATP, acetyl CoA, and NADH are allosteric repressors. They inactivate pyruvate dehydrogenase. When concentrations of these are high, the cell has plenty of energy (and isn't using it all up) so no more ATP is needed D. The citric acid cycle (also known as the Krebs Cycle) The acetate part (two carbons) of acetyl CoA is transferred to oxaloacetate (a four carbon compound) to form citrate (a six carbon compound) and the citric acid cycle has begun! * * * * * * * * * * 97 C. The major points of the citric acid cycle 1) The pathway is a cycle! Two carbons come in (from acetate), two carbons are released as 2 CO ,2and the four carbon compound oxaloacetate is reformed, ready for entry of another acetate 2) One ATP is formed per cycle 3) Three NADH are reduced from three NAD+ 4) One FAD is reduced to FADH2 FAD [flavin adenine nucleotide] is an oxidizing agent just like NAD . It can oxidize compounds and get reduced to FADH 2 ***** Remember, each glucose produces two pyruvate. So the complete degradation of 98 glucose requires two cycles through the pathway (one cycle for each of the acetates that are derived from the two pyruvates) 5) The chemical equation for each cycle of the citric acid cycle is: Acetyl CoA + 3 NAD+ + FAD + ADP + Pi 2 CO2 + CoA + 3 NADH + FADH2 + ATP 6) Starting with glucose, and going twice through the citric acid cycle gives: + Glucose + 10 NAD + 2 FAD + 4 ADP + 4 Pi 6 CO + 10 NAD2 + 2 FADH + 4 ATP 2 (six carbons) (six carbons) So far, the degradation of glucose has yielded 4 ATP by substrate level phosphorylation Each NADH will be oxidized to NAD yielding 3 ATP (its 2.5 to 3) Each FADH will be oxidized to FAD yielding 2 ATP (its about 1.8 to 2) 2 (its easier to use whole numbers so we (and nearly everyone else) uses the value 3 and 2) Together, oxidation of the 10 NADH and the 2 FADH yields234 ATP So, the complete degradation of glucose will yields 38 ATP. However, moving the two NADH from the cytoplasm to the mitochondria where they get oxidized costs one ATP each So, the total net ATP made from oxidizing one molecule of glucose under aerobic conditions is 36! D. Other important points of the citric acid cycle: 1) Glycolysis takes place in the cytosol 2) In eukaryotes, the citric acid cycle takes place in the mitochondria (in bacteria, because there are no membrane bounded organelles, the citric acid cycle takes place, like glycolysis, in the cytosol) 3) The enzyme pyruvate dehydrogenase complex is in the mitochondria. So pyruvate must get from the cytosol to the mitochondria 4) Pyruvate diffuses through the mitochondrial membrane so that it can be converted into acetyl CoA by pyruvate dehydrogenase. Lecture 16- The Respiratory Chain 99 From glycolysis and the citric acid cycle, only 4 ATP are produced from one molecule of glucose. Most of the energy derived from oxidizing glucose to 6 CO was used2to + reduce NAD and FAD to NADH and FADH . 2 The reoxidation of NADH and FADH by the r2spiratory chain leads to the majority of ATP produced when glucose is oxidized in organisms that can use oxygen A. The Respiratory Chain The respiratory chain is a series of four protein complexes and one organic molecule (coenzyme Q) that are embedded in the inner membrane of mitochondria (in eukaryotes) or the plasma membrane (in prokaryotes) The enzymes associate with each other and function to transport electrons, released from the oxidation of NADH and FADH2 ***The respiratory chain is a series of oxidationreduction reactions The final acceptor for the electrons is O w2ich gets reduced to H O 2 This process releases a lot of energy which is used to generate ATP from ADP The drop in energy of the electrons starting with NADH and ending with the transfer of electrons 2 (with two H to form H O i2 53 kcals/mol. It is this energy that is used to produce ATP by to O oxidative phosphorylation ***When NADH transfers its electrons, it gets oxidized to NAD +. NAD can now participate in glycolysis and the citric acid cycle and get reduced again when another molecule of glucose gets oxidized It takes 2 NADH to reduce one molecule of O to 2 H2O 2 The proteins' amino acid chains are not getting reduced. These proteins have cofactors that get reduced Coenzyme Q both accepts electrons (getting reduced) and picks up protons which it transfers to the other side of the membrane Two electrons going down the chain is very exergonic. Enough energy is + released to pump about 20 H across the membrane There are 26 polypeptides in the protein complex that accepts electrons from NADH Cyanide is an inhibitor of Cyt C. Carbon monoxide is an inhibitor (competitive or noncompetitive?) of the enzyme that reduces oxygen to water B. How ATP is generated by electrons passing through this chain Transfer of the electrons along the respiratory chain results in the generation of a proton (H ) gradient (proton motive force) At each step in the chain there is sufficient energy released to drive H against the concentration gradient (remember, this requires energy) from the matrix of the 100 mitochondria into the inner membrane space of the mitochondria + The generation of the proton gradient, with a super high concentration of H in the inner membrane space and a low concentration of H+ in the matrix of the mitochondrion, provides the energy to ATP synthase to convert ADP into ATP, using the flow of H+ back into the matrix through ATP synthase. This theory of how ATP is generated is called the chemiosmotic theory Together the 10 NADH and 2 FADH2 produced from oxidizing glucose result in 34 ATP made by oxidative phosphorylation C. Compounds that uncouple the respiratory chain from ATP synthesis There are compounds that uncouple ATP synthesis from the oxidation/reduction reactions + These compounds work by allowing the free flow of H back across the inner membrane into the matrix without going through the ATP synthase enzyme H is pumped, but no ATP is made. The energy normally used to make ATP dissipates as heat Some animals that live in very cold climates produce natural uncouplers (thermogenin) so that heat can be produced instead of ATP. Dinitrophenol is also an uncoupler. Uncouplers are weak acids that can penetrate the + mitochondrial intermembrane space and carry H back across the membrane. No more proton motive force. NADH is still oxidized to NAD ! Glycolysis can continue! D. Other compounds that have profound effects of the respiratory chain Cyanide and arsenic. These compounds are extremely toxic because they shut down both electron transfer and ATP synthesis. 2 ATP 34 ADP 34 ATP 36 ATP Lecture 17- Fermentation 101 A. Fermentation Fermentation is the anaerobic breakdown of carbohydrates In cells with an insufficient supply of oxygen, there is no O to2be the final acceptor of electrons in the respiratory chain. The respiratory cha+n shuts down. NADH and FADH ca2not be oxidized and so there is no NAD or FAD for the citric acid cycle Without the respiratory chain, pyruvate does not get converted into acetyl CoA and the cell is completely dependent on glycolysis for the production of ATP Glycolysis "use up" NAD+ during the step when it is reduced to NADH (step 6 of glycolysis) If the cell cannot reform NAD + for use in this step, glycolysis will shut down and the cell will die from lack of ATP production + Muscle cells have the ability to reform NAD These cells have an enzyme, lactate dehydrogenase, that reduces pyruvate to lactate and at the same time, oxidizes NADH (produced during glycolysis) to + NAD . This is referred to as lactate (or lactic acid) fermentation Muscle cells can therefore keep glycolysis going in the absence of oxygen Strenuous exercise depletes your system of oxygen respiration switches from aerobic (able to make ATP through oxidative phosphorylation) to anaerobic (pyruvate lactate) Anaerobic respiration is much less efficient! Only glycolysis is taking place and only 2 ATP are produced when 1 molecule of glucose is degraded to two molecules of lactate (the final product) During anaerobic respiration, lactate build up in muscles Lactate is toxic The build up of lactate causes leg cramps because of its toxicity When glucose is all used up, triglycerides (glycerol + 3 fatty acids) are used as fuel. The fatty acids get broken down to acetyl units of acetyl CoA, which go through the citric acid cycle. The glycerol gets broken down into an intermediate of glycolysis and gets converted into pyruvate and then acetyl CoA 102 B. Anaerobic respiration in prokaryotes Some anaerobic bacteria also degrade glucose to lactate, and produce ATP only through glycolysis Some anaerobic bacteria also have a respiratory chain, but use nitrogen or sulfur as the final electron acceptor (bacteria that live miles below the surface only use sulfur since O not present). 2 If life exists on an ammonium (not water) based world, O coul2 not be the final electron acceptor, it would have to be N NH 2 3 C. Anaerobic respiration in yeast Yeast are single cell eukaryotes Yeast can grow either aerobically or anaerobically Yeast growing anaerobically do not produce lactate from pyruvate. Instead, they undergo alcohol fermentation In alcohol fermentation, a carbon is removed from pyruvate (as CO ) leavi2g the two carbon compound acetaldehyde Acetaldehyde is reduced to ethanol and NADH is oxidized to NAD+ Lecture 18- Additional biochemical pathways A. Fats and proteins break down into acetyl CoA and intermediates in glycolysis and citric acid cycle. All 20 amino acids have their own breakdown pathways 103 The breakdown products are also the products used to assemble complex molecules. But remember, the breakdown and biosynthetic pathways cannot be the same. Fatathway for Phospholi synthesis pids s of ribose Lecture 19- Photosynthesis A. Some basic facts of life Heterotrophs must eat organic molecules (like sugars, proteins) to live Autotrophs can form all of their organic molecules from inorganic molecules Autotrophs use the energy of the sun to make sugars (glucose) from the carbons in CO 2nd the hydrogens and oxygens come from H 0. 2rom glucose, all other sugars can be made. Autotrophs are crucial for heterotrophs! 104 Glucose CO G = 2 kcals/mole (262 kcals/mole used to make ATP) CO 2Glucose G = +686 kcals/mole Energy from the sun is used to make ATP and reduce NADP to NADPH+ That ATP supplies the energy to make glucose from CO 2 NADPH supplies the hydrogens in glucose. The plant can then use each glucose it just made to synthesize 36 ATP to use to run its cells. B. The basic reactions of photosynthesis Photosynthesis is the conversion of light energy (photons) into chemical energy (covalent bonds). The products of photosynthesis are glucose (and other sugars) and oxygen Photosynthesis takes place in chloroplasts The enzymes required are embedded in the thylakoid membrane (th
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