1A. Hint: What pathways are involved here? Do you have the enzymes for Krebs etc.? In other words, what is DHAP being converted to? When you figure out the overall pathway, look carefully at the stoichiometry -- for every DHAP that goes through, what compounds are used up, and what compounds are synthesized (or regenerated)?

    Explanation: If you check out the stoichiometry of the pathway, you see that two ADP's are phosphorylated to ATP per DHAP broken down. No input of ATP is needed; you have bypassed the steps in glycolysis that use ATP by starting with DHAP instead of glucose. (When you start with glucose, you have to put in 2 ATP to "prime the pump." When you start with DHAP, you don't need to "spend" any ATP to get started.)  For each DHAP, one inorganic phosphate is picked up from solution, to make a di-phosphorylated intermediate. The two phosphates are then transferred in two separate steps to phosphorylate two ADP's to ATP. No CoA is involved -- it is needed for entry into the Krebs cycle, which is not occurring here. One NAD is needed for oxidation of each DHAP for step 6 of glycolysis. However the NAD reduced to NADH₂ in step 6 is recycled -- it is oxidized back to NAD in the last step of fermentation. So you only need a catalytic amount of NAD.

1B. Hint: Look at your handout (7-2) -- label the proper carbon in DHAP and trace it through the pathway. (What will be the end product of the pathway here?) What happens when you convert DHAP to glyceraldehyde -3-P ? Does the P move, or do you flip the molecule over -- so the "top" carbon in DHAP becomes the "bottom" carbon in glyceraldehyde -3-P?

    Answer: Ethanol. The #1 carbon of DHAP (top as written on handout 7-2)  becomes #3 carbon  of glyceraldehyde-3 phosphate (bottom as written on handout). The phosphate group doesn't move -- you flip the DHAP molecule over and the hydroxyl on top becomes a carbonyl and the carbonyl at position 2 becomes a hydroxyl. The labeled carbon (now #3) becomes the methyl group of pyruvate. The pyruvate is subsequently decarboxylated to yield acetaldehyde and then reduced to ethanol in the ethanolic fermentation found in yeast. (If you forgot to flip the DHAP, then you would think that the radioactivity would be lost as CO₂.) Next best answer is lactate, if you forgot that yeast do ethanolic and not lactate fermentation. Third best answer is pyruvate, if you mistakenly added more than the minimum NAD to the reaction, such that fermentation is not necessary (though it would presumably take place anyway). Acetyl CoA would not be formed, as only the glycolytic and fermentation enzymes are present. The other compounds have no connection to this carbon.

1C. Hint: Consider the yields of ATP and the NAD_red/NAD_ox balance. What conditions must be met for the bacteria to grow?

    Answer: yes. There is a net gain of ATP, and no net oxidation or reduction of NAD -- what is used is regenerated. Only one NAD is necessary in the glycolytic pathway converting DHAP to pyruvate, and that one NAD can be regenerated from NADH₂ in the final step of fermentation, the reduction of pyruvate to lactate (the usual product in E. coli).

Note:  E. coli can't really live on DHAP because phosphorylated sugars can't get transported into the cells.

2. A. Hint: Will FAD donate electrons/hydrogens to NAD, or vice versa? Consider where the two electron carriers normally feed into the electron transport chain.

    Answer: Positive.  There are several ways to see this:

    (i) By looking at the order in the electron transport chain: Reduced FAD feeds into the chain after reduced NAD -- that is, below it, on an energy scale. (See handout 8-2.) So NADH₂  should hand off hydrogens and/or electrons to FAD and not the other way around, since electrons go down the chain, not up it. In terms of affinity for electrons, the compounds "higher up" on the chain have a lower affinity for electrons, and tend to give them away to compounds further down the chain.  The compounds "lower down" on the chain have a higher affinity for electrons, and tend to grab electrons from the compounds above. (Oxygen, which is on the bottom, has the highest affinity for electrons -- it gets them in the end.)
    In terms of  ΔG^o: if NADH₂ is "higher up" on the chain, then the distance from it to oxygen is greater, and so the ΔG^o for reduction of NADH₂ must be more negative (than for FADH₂). If you want to formally add up the ΔG^o values for oxidation/reduction of NAD and FAD to get the overall ΔG^o for the reaction shown in the problem, see (iii).

    (ii) By considering relative ATP yields -- Another way to get the relative ΔG^o values: Oxidation of FADH₂ drives production of only 2 ATP's while oxidation of NADH₂  drives production of 3 ATP's. So you must get more energy from oxidation of reduced NAD, so the ΔG^o for reduction of NADH₂ must be more negative.

    (iii) Adding up the ΔG^o values: 
   
            NADH + H⁺ + 1/2 O₂ ↔ NAD + H₂O   ΔG^o = -52 kc/mole or some large negative number if you don't remember exactly.

  FADH₂ + 1/2 O₂ ↔ FAD + H₂O  ΔG^o = a much smaller negative number for the reasons given in (i) & (ii) -- since FAD enters the electron transport chain at a lower level, and since one derives less ATPs/electron pair from FADH₂ than from NADH + H⁺.

    Inverting the top reaction and changing the sign:

    NAD  ↔ NADH + H⁺ + 1/2 O₂  ΔG^o = +52 kc/m or some large positive number

    FADH₂ + 1/2 O₂ ↔ FAD + H₂O  ΔG^o = a much smaller negative number

    Net: NAD + FADH₂ ↔ FAD + NADH + H⁺  ΔG^o =  a large positive number plus a much smaller negative number = highly positive.

2B. Hint: Where will the electrons/hydrogens of reduced NAD go in the bacteria? Will they follow their usual path?

    Answer: Less. In the bacteria, because there is so much enzyme present, all the NADH₂ will give up its electrons to FAD and then the FADH₂ will enter the electron transport chain "lower down" bypassing the first site of ATP formation. In humans, the NADH₂ will give up its electrons directly to the electron transport chain -- it will enter "higher up," before the first site of ATP synthesis. Therefore, in the bacteria, the electrons from  NADH₂ will only yield 2 ATP, compared to the 3 ATP/NADH₂ in humans. Since there are 10 NADH₂'s produced from the oxidation of one molecule of glucose, the difference in ATP yield is considerable. In bacteria, the yield is 10 X 2 = 20 ATP vs. 30 ATP in humans, a difference of 10 less in the bacteria.

 2C.   Hint: Will the bacteria get anything out of this reaction? (Where do you think these bacteria were found?) Where does the energy normally used to make ATP go?

    Answer: Such an enzyme would generate heat by 'wasting' energy that would otherwise be used to drive electron transport and ATP synthesis. Although this example is made up, many organisms do use exergonic reactions for heat generation under specific circumstances.

3. A. Hints:
    (i). If you have a piece of inner membrane in its normal place, and you have the compounds listed, what processes will take place? Ox. phos? Electron transport? Krebs cycle? Fermentation? Anything else?

    (ii). In the apparatus, one side (left or right) is equivalent topologically to the mitochondrial matrix, and one side is equivalent topologically to the space between the mitochondrial membranes. Which is which? Once you've figured that out, where do electrons & protons normally go during electron transport, ox. phos., etc.? What chemical reactions involving NAD, ATP etc. normally occur on each side of the membrane?

3A. Answers: 

    A-1. pH will increase in the left. Hydrogen ions will be pumped across the membrane in the direction from what had been the inside or matrix side (left) to what had been the intramembrane space (right). The pH on the right will fall and the pH on the left will rise. (The hydrogen ion concentration will increase on the right and fall on the left.)

    A-2. Increase in the left. ATP will be synthesized from ADP + P_i as hydrogen ions flow back across the membrane from right to left through the ATP synthetase of the F_oF₁ complex in the membrane.

    A-3. NAD_ox will increase on the left. The reduced NAD gives up its electrons to the electron transport chain in the membrane, interacting with components on the inside or matrix side (left) of the membrane. (The electron chain is embedded asymmetrically in the membrane, so the protein complex that binds NAD faces the matrix side.)

    A-4. Neither. The reduced NAD (NAD_red) will decrease on the left as it gives up its electrons to the electron transport chain and is converted to NAD_ox in the membrane.

    A-5. Neither. Pyruvate can be metabolized two ways in a cell, but neither can occur here. Pyruvate can be metabolized by the entrance reaction to the Krebs Cycle, which requires CoA (Coenzyme A) to run, and CoA is not included (nor are the enzymes of the matrix). Pyruvate can also be metabolized by conversion to lactic acid in fermentation, but the enzymes to do this are found in the cytoplasm, and are not included here.

    B. Hint: Which of the processes taking place require oxygen?

    Answers: pH, ATP, and NAD_ox or NADH_red. If there is no O₂ present to accept the electrons for the electron transport chain, then the electron transport chain will not be able to accept the electrons from NAD_red. So compared to the situation above, there will be less NAD_ox  and more NAD_red on the left.  If there is no electron transport, no hydrogen ions will be pumped across the membrane, and no hydrogen ion gradient will be established. Therefore there will be no net flow of hydrogen ions back through the ATP synthetase and no ATP production (on the left). 

    C.  Hint: Why do you normally need to maintain different conditions on the two sides? 

    Answers: Phosphorylation of ADP to ATP & establishment of a pH difference between the two chambers will be affected.

The electron transport chain will continue to run and to pump hydrogen ions from left to right across the membrane, but hydrogen ions will not accumulate in the right chamber since the pumped-out hydrogen ions will return to the left chamber through the tear in the membrane. Thus no pH gradient will be established. With no pH gradient, there will be no flow of hydrogen ions though the ATP synthetase, and no ATP will be produced.
    NAD oxidation (oxidation of NAD_red to NAD_ox) will continue using the electron transport chain components in the membrane. The ETC components are located asymmetrically in the membrane, so NAD  oxidation will occur on the left side only, as before. (You have uncoupled ox. phos. from electron transport.) However, some of the NAD_ox generated on the left will diffuse into the right compartment through the tear, and some of the NAD_red will diffuse from right to left, reducing the difference in NAD_ox  and NAD_red between the two sides. NAD oxidation may be slower, but it will continue.