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III. Glycolysis

  1. Introduction
    1. Glycolysis = (Glyco-sweet, sugar; lysis=to split) Metabolic pathway where six-carbon glucose is split into two three-carbon sugars which are then oxidized and rearranged by a step-wise process producing two pyruvic acid molecules.
      1. Occurs in all cells.
      2. As the two intermediate three-carbon sugars are converted to pyruvic acid:
        1. Two molecules of NAD+ are reduced to NADH.
        2. There is a net production of two ATPs by substrate-level phosphorylation.
      3. The ten steps of glycolysis occur in the cytoplasm and are catalyzed by specific enzymes, dissolved in the cytosol. These steps can be grouped into two segments:
        1. Preparatory steps.
        2. Oxidative steps.
  2. The Steps in Glycolysis: You do not have to memorize these reactions; but, you must understand the overall reaction.
    1. The first phase includes five preparatory steps where glucose is split in two. This process actually consumes ATP.
    2. Step 1: Glucose enters the cell and is phosphorylated on the number six carbon. This ATP coupled reaction:
      1. Is catalyzed by hexokinase. (Kinase is an enzyme involved in phosphate transfer.)
      2. Requires an initial investment of ATP.
      3. Makes glucose more chemically reactive.
      4. Produces glucose-6-phosphate. Since the plasma membrane is relatively impermeable to ions, the addition of an electrically charged phosphate group traps the sugar in the cell
    3. Step 2: An isomerase catalyzes the rearrangement of glucose-6-phosphate to its isomer, fructose-6-phosphate.
    4. Step 3: Carbon one of fructose-6-phosphate is phosphorylated. This reaction:
      1. Required an investment of still another ATP.
      2. Is catalyzed by phosphofructokinase, an allosteric enzyme that controls the rate of glycolysis.
    5. Step 4: Aldolase cleaves the six-carbon sugar into two isomeric three-carbon sugars.
      1. This is the reaction for which glycolysis is named.
      2. For each glucose molecule that begins glycolysis, there are two product molecules for this and each succeeding step.
    6. Step 5: An isomerase catalyzes the reversible conversion between the two three-carbon sugars. This reaction:
      1. Never reaches equilibrium because only one isomer, glyceraldehyde phosphate, is used in the next step of glycolysis.
      2. Is thus pulled towards the direction of glyceraldehyde phosphate, which is removed as fast as it forms.
      3. Results in the net effect that, for each glucose molecule, two molecules of glyceraldehyde phosphate progress through glycolysis.
    7. The second segment of reactions is where oxidation of the sugar occurs. These last five steps produce ATP and NADH.
    8. Step 6: An enzyme catalyzes two sequential reactions:
      1. Glyceraldehyde phosphate is oxidized and NAD+ is reduced to NADH + H+.
        1. This reaction is very exergonic (Delta G = -10.3 kcal/mol) and is coupled to the endergonic phosphorylation phase.
        2. For every glucose molecule, 2 NADH are produced.
      2. Glyceraldehyde phosphate is phosphorylated on carbon number one.
        1. The phosphate source is inorganic phosphate, which is always present in the cytosol.
        2. The new phosphate bond is a high energy bond at least as energetic as the phosphate bonds of ATP.
    9. Step 7: ATP is produced by substrate level phosphorylation.
      1. In a very exergonic reaction, the phosphate group with the high energy bond is transferred from 1, 3-diphosphoglyceric acid to ADP.
      2. For each glucose molecule, two ATP molecules are produced. The ATP ledger now stands at zero as the initial debt of two ATP from steps one and three is repaid.
    10. Step 8:
      1. In preparation for the next reaction, a phosphate group on carbon three is enzymatically transferred to carbon two.
    11. Step 9: Enzymatic removal of a water molecule:
      1. Creates a double bond between carbons one and two of the substrate.
      2. Rearranges the substrate's electrons, which transforms the remaining phosphate bond into an unstable high energy bond.
    12. Step 10: In this last step of glycolysis, ATP is produced by substrate level phosphorylation.
      1. In a highly exergonic reaction, a phosphate group is transferred from PEP to ADP.
      2. For each glucose molecule, this step produces two ATP.
    13. Summary of Glycolysis:

      The summary equation for glycolysis is:

      C6H12O6 + 2 NAD+ + 2 ADP + 2 P -->
      2 C3H4O3 (Pyruvic Acid) + 2 NADH + 2 H+ + 2 ATP + 2 H2O

       

      1. Glucose has been broken down and oxidized into two pyruvic acid molecules.
      2. The process is exergonic (Delta-G = -140 kcal/mol), and most of the energy made available is conserved in the high-energy electrons of NADH and the phosphate bonds of ATP.
      3. The first segment of glycolysis uses two ATP per glucose molecule.
      4. The second segment of glycolysis produces four ATP per glucose molecule, which is a net gain of two ATP per glucose. These ATP are produced by substrate-level phosphorylation.
      5. Glycolysis produces two molecules of NADH per glucose. Energy conserved in the high-energy electrons of NADH can be used later to make ATP by oxidative phosphorylation.
      6. Most of the chemical energy originally stored in glucose still resides in the two pyruvic acid molecules produced by glycolysis.

      7. V. THE ELECTRON TRANSPORT CHAIN AND OXIDATIVE PHOSPHORYLATION

        1. Introduction
          1. The electron transport chain:
            1. Is made of electron carriers embedded in the inner mitochondrial membrane.
            2. Passes electrons from reduced coenzyme (NADH and FADH2) to oxygen.
            3. The Krebs Cycle occurs only under aerobic conditions, it does not use oxygen directly. The electron transport chain and oxidative phosphorylation require oxygen.)
            4. Uses electron flow to create a proton gradient across the inner mitochondrial membrane.
          2. Oxidative phosphorylation is coupled to the electron transport chain by the potential energy stored in the proton gradient.
            1. ATP synthase catalyzes the phosphorylation of ADP. The energy required to power this process is released when protons diffuse down the gradient through the enzyme complex.
            2. The overall function of oxidative phosphorylation is to transform electron energy of glucose into energy stored in the phosphate bonds of ATP.
        2. Electron carrier molecules transfer electrons down the electron transport chain.
          1. Prosthetic groups of electron carriers shift between reduced and oxidized states as they accept and donate electrons.
          2. Most electron carriers are proteins. Ubiquinone (Q) is the only electron carrier of the transport chain that is not bound to a protein.
          3. Electron carriers of the transport chain, which are proteins include:

            Protein Electron Carriers

            Prosthetic Group

            flavoprotein

            flavin mononucleotide (FMN)

            iron-sulfur protein

            iron and sulfur

            cytochromes

            heme group

          4. Heme group = Prosthetic group composed of four organic rings surrounding a single iron atom.
          5. Cytochromes = Type of protein molecule which contains a heme prosthetic group and which functions as an electron carrier in the electron transport chains of mitochondria and chloroplasts.
          6. There are several types of cytochromes, each a different protein with a heme group.
          7. It is the iron of cytochromes that transfers electrons.
          8. Sequence of Electron Transfers Along the Electron Transport Chain:
          9. Electron transfer from NADH to oxygen is exergonic (Delta-G = -53 kcal/mol).
          10. Free energy change does not occur in one explosive step. Instead, electrons lose a small amount of energy as they cascade down the chain from carrier to carrier.
          11. Oxygen, the terminal electron acceptor, has a great affinity for electrons and pulls electrons down the chain.
          12. FADH2 also donates electrons to the electron transport chain, but those electrons are added at a lower energy level than NADH.
          13. The electron transport chain does not make ATP directly. It generates a proton gradient across the mitochondrial membrane, which stores potential energy that can be used to phosphorylate ADP.
        3. Generation of the Proton Gradient
          1. The electron transport chain generates a proton gradient by transporting protons from the mitochondrial matrix to the intermembranal space.
            1. Some electron carriers of the transport chain accept and release protons along with the electrons.
            2. Other carriers transport only electrons.
          2. Proton translocation is based on spatial organization of the electron transport chain in the membrane.
          3. Electron carriers are organized into three complexes:
            1. NADH dehydrogenase complex.
            2. When reduced, the complex also accept two protons from the matrix (one from NADH and one from solution).
            3. When oxidized, the complexes passes on electrons and also releases two protons into the intermembrane space.
            4. Cytochrome b-c1 complex.
            5. This complex may translocate additional protons from the matrix to the intermembrane space.
            6. It passes electrons to the mobile carrier, cytochrome c.
            7. Cytochrome oxidase complex.
            8. This complex passes electrons to oxygen.
          4. Mobile carriers transfer electrons between complexes. These mobile carriers are:
            1. Ubiquinone (Q).
              1. Near the matrix, Q accepts electrons from the NADH dehydrogenase complex and two protons from solution.
              2. Q diffuses across the lipid bilayer and releases two protons to the intermembrane space.
              3. As protons are released, Q passes electrons to the cytochrome b- c1 complex.
            2. Cytochrome c (Cyt c).
              1. Cyt c is reduced as it accepts electrons from the cytochrome b-c1 complex.
              2. As it is oxidized, Cyt c conveys its electrons to the cytochrome oxidase complex.
          5. When the transport chain is operating:
            1. The pH in the intermembrane space is one or two pH units lower than in the matrix.
            2. The pH in the intermembrane space is the same as the pH of the cytosol because the outer mitochondrial membrane is permeable to protons.
        4. The Proton-Motive Force and ATP Synthesis
          1. Proton motive force = Potential energy stored in the proton gradient created across biological membranes that are involved in chemiosmosis.
            1. This force is an electrochemical gradient with two components:
              1. Concentration gradient of protons (chemical gradient).
              2. Voltage across the membrane because of a higher concentration of positively charged protons on one side (electrical gradient).
            2. It tends to drive protons across the membrane back into the matrix, but the inner mitochondrial membrane is not very permeable to protons.
            3. Protons reenter the matrix by passing through an ATP-synthesizing protein complex that spans the inner mitochondrial membrane. This complex is ATP synthase.
          2. Multiple copies of ATP synthase stud the inner mitochondrial membrane. They function to couple the exergonic passage of protons with the endergonic phosphorylation of ADP.
          3. This complex of several proteins has two main components:
            1. F0 - This part spans the membrane and channels proton diffusion.
            2. F1 - Attached to F0 on the matrix side of the inner mitochondrial membrane, this spherical part catalyzes ADP phosphorylation. How the F1 enzyme uses energy from the proton current is still unknown.
          4. Effects of three classes of respiratory poisons provide evidence for chemiosmosis and its dependence upon the structural organization of the mitochondrial membrane.
            1. Poisons that block electron flow.
              1. Cyanide blocks electron flow from Cyta3 to oxygen. This stops the electron transport chain so it cannot pump protons. Without the proton gradient, ATP is not produced.
            2. Poisons that make the inner mitochondrial membrane leaky to protons.
              1. These poisons, such as dinitrophenol, are called uncouplers. By allowing protons to leak back across the membrane, they uncouple the process of proton pumping with ATP production.
            3. Poisons that inhibit ATP synthase.
              1. This class of poisons, which includes the antibiotic oligomycin, directly inhibits ATP synthase.
              2. The proton gradient becomes greater than normal and yet the potential energy of the gradient cannot be tapped to produce ATP.
        5. The ATP Ledger for Respiration
          1. The net ATP yield from the oxidation of a glucose molecule is influenced by several factors:
            1. For each high energy electron pair that travels from NADH down the electron transport chain to oxygen, enough proton-motive force is created to produce a maximum of three ATPs.
            2. FADH2 is worth a maximum of only two ATPs, since it donates electrons at a lower energy level to the electron transport chain.
            3. In most eukaryotic cells, the ATP yield is lower from an NADH produced during glycolysis. The mitochondrial membrane is impermeable to NADH, so its electrons must be shuttled across the membrane. These electrons are received inside the mitochondrion by FAD, a process which downgrades the energy level of those electrons.
            4. There is a debit of two ATPs from the preparatory steps of glycolysis, and everything is doubled after the sugar-splitting step of glycolysis.
            5. This tally only estimates the ATP yield from respiration. Some variables that affect ATP yield include:
              1. Mitochondrial membranes may differ in permeability to protons.
              2. The proton motive force may be used to drive other kinds of work such as active transport.
              3. The estimate of 36 ATPs produced per glucose is contingent upon an adequate oxygen supply.
              4.  

                I. FERMENTATION: THE ANAEROBIC ALTERNATIVE

                1. Introduction: Some definitions
                  1. Aerobic = Existing in the presence of oxygen.
                  2. Anaerobic = Existing in the absence of free oxygen.
                  3. Fermentation = The anaerobic catabolism of organic nutrients.
                2. Glycolysis oxidizes glucose to two pyruvic acid molecules. The oxidizing agent for this process is NAD+, not oxygen.
                  1. Some energy released from the exergonic process of glycolysis drives the production of 2 ATPs (net) by substrate-level phosphorylation.
                  2. Glycolysis produces a net of 2 ATPs whether conditions are aerobic or anaerobic.
                    1. Aerobic conditions: Pyruvic acid is oxidized further, and more ATP is made as NADH gasses electrons removed from glucose to the electron transport chain. NAD+ is regenerated in the process.
                    2. Anaerobic conditions: Pyruvic acid is reduced, and NAD+ is regenerated. This prevents the cell from depleting the pool of NAD+ which is the oxidizing agent necessary for glycolysis to continue. No additional ATP is produced.
                3. Fermentation consists of anaerobic glycolysis plus subsequent reactions that regenerate NAD+ by reducing pyruvic acid. Two of the most common types of fermentation are:
                  1. Alcohol Fermentation
                    1. Pyruvic acid is converted to ethanol in two steps:
                      1. Pyruvic acid loses carbon dioxide and is converted to the two- carbon compound acetaldehyde.
                      2. NADH is oxidized to NAD+ and acetaldehyde is reduced to ethanol.
                    2. Many bacteria and yeast carry out alcohol fermentation under anaerobic conditions.
                  2. Lactic Acid Fermentation
                    1. NADH is oxidized to NAD+ and pyruvic acid is reduced to lactic acid.
                      1. Commercially important products of lactic acid fermentation include cheese, yogurt, acetone and methyl alcohol.
                      2. When oxygen is scarce, human muscle cells switch from aerobic respiration to lactic acid fermentation. Lactic acid accumulates, but it is gradually carried to the liver where it is converted back to pyruvic acid when oxygen becomes available.
                      3.            

                        VII. COMPARISON OF AEROBIC AND ANAEROBIC CATABOLISM

                        1. Introduction
                          1. There are three major catabolic processes for harvesting food's chemical energy
                            1.  
                            2. aerobic respiration
                            3. anaerobic respiration
                            4. fermentation.
                          2. These processes:
                            1. Are similar in that the high energy electrons from substrate (e.g. glucose) oxidation are transferred to NAD+.
                            2. Differ in the ultimate fate of high energy electrons stored in NADH.
                        2. Cellular respiration includes all types of catabolism that use electron transport chains to make ATP, regardless of the substance used as the final electron acceptor. It also includes the Krebs cycle or some modification of the Krebs cycle.
                        3. Aerobic respiration occurs only in the presence of oxygen. This process:
                          1. Uses an electron transport chain to make ATP.
                          2. Uses oxygen as the final electron acceptor.
                          3. Produces most ATP by oxidative phosphorylation and some ATP by substrate-level phosphorylation.
                          4. Is used by plants and animals.
                        4. Anaerobic respiration is a catabolic process that does not require the presence of oxygen. This process:
                          1. Uses an electron transport chain to make ATP.
                          2. Uses a substance other than free oxygen as a final electron acceptor (e.g. NO3-, SO42-, or CO32-).
                          3. Produces most ATP by oxidative phosphorylation and some by substrate-level phosphorylation.
                          4. Occurs in a few bacterial groups that exist in anaerobic environments.
                        5. Fermentation operates not only without electron transport chains, but without the Krebs cycle as well.
                          1. No oxygen is required.
                          2. The final electron acceptor is an organic substrate such as pyruvic acid or some derivative of pyruvic acid.
                          3. ATP is produced by substrate level phosphorylation only.
                          4. This process is less efficient than respiration. Respiration yields 18 times more ATP per glucose than fermentation.
                        6. Organisms can be classified based upon the effect oxygen has on growth and metabolism.
                          1. Strict (obligate) aerobes = Organisms that require oxygen for growth and as the final electron acceptor for aerobic respiration.
                          2. Strict (obligate) anaerobes = Microorganisms that only grow in the absence of oxygen and are, in fact, poisoned by it.
                          3. Facultative anaerobes = Organisms capable of growth in either aerobic or anaerobic environments.
                            1. Yeasts, many bacteria and mammalian muscle cells are facultative anaerobes.
                            2. Some facultative anaerobes make ATP only by fermentation in the absence of oxygen, and make ATP by respiration in presence of oxygen.
                            3. Glycolysis is common to fermentation and respiration. In some facultative anaerobes, the fate of pyruvic acid will be either fermentation or respiration depending upon the presence of absence of oxygen.
                        7. Evolutionary Significance of Glycolysis:
                          1. The first prokaryotes probably produced ATP by glycolysis. Evidence includes the following:
                            1. Glycolysis does not require oxygen, and the oldest known bacterial fossils date back to three-and-a-half billion years ago when oxygen was not present in the atmosphere.
                            2. Glycolysis is the most widespread metabolic pathway, so it probably evolved early.
                            3. Glycolysis occurs in the cytoplasm and does not require membrane- bound organelles. Eukaryotic cells with organelles probably evolved about two billion years after prokaryotic cells
 
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