BIOCHEMISTRY

Biochemistry

Exam II

24 February 2004

A certain type of cells which we will call BTQ cells are known to be able to convert the common compound A into the required synthetic precursor, B. An extract from these cells, properly prepared, is able to catalyze this overall transformation. If compound X is added, B is not made, but another compound, C, accumulates. A mutant line of cells, BTQ-M, cannot make B from A, but can make B if it is provided with D in the medium. In the presence of X, BTQ-M cannot make B even if provided with D. A second mutant, BTQ-N, also fails to grow on A, but can grow if provided with B, or alternatively, with E. It is unable to use D. In the presence of X, it can still make B from E. What conclusions can you draw about the pathway, A ->B, the mutants M and N, and the role of X?
In mitochondria, complex III has three interactions with quinone (CoQ). What are they? What would be the effect on the electron transport chain of inhibiting each of these three interactions one at a time, in the presence of succinate, O₂, ADP and P_i? Adding ascorbic acid to isolated mitochondria will feed electrons directly to cytochrome c. How does an excess of ascorbic acid affect the redox state of the mitochondrial electron carriers in the absence of O₂? Which carriers are not affected and why? Now add antimycin (blocks at complex III) and succinate; are any other redox carriers affected? In fact, it turns out that in this system the b cytochromes remain oxidized. Explain. Finally, O₂ is added. Now, cyt c and c₁ get oxidized, and the b cytochromes get reduced. Explain. What step is antimycin blocking?
Thiamine is known to catalyze the conversion of pyruvate to acetoin. Suggest a mechanism for this reaction.
The F_o portion of the coupling factor contains a proton channel, which requires both the c-ring and the a subunit. We introduce the following mutations: in the c subunit, asp61 is changed to lysine, in the a subunit, arg210 is changed to glutamate. We find that the maximal rate of ATP synthesis is greatly reduced. What will happen to the rate of ATP hydrolysis? Why? We try again. The c subunit asp61 is changed to histidine. The a subunit arg 210 is changed again to glutamate. The maximal rate of ATP synthesis is only slightly reduced from wild type. Why? Would these mutations have any effect on the ability of F_o to conduct protons, by itself? Is the direction of rotation of the c-ring important? If so, why, and what determines this direction?
Some recent studies on glucokinase in the liver have produced interesting results. For instance, it is found that the flux control coefficient for glucokinase in the synthesis of glycogen from glucose is quite large. The flux control coefficient for GKRP in glycogen synthesis is near –1. What do these results mean? It turns out that C^J for GKRP in glycolysis is about –0.5. What does this suggest regulates glycolytic flux? What do you expect to be the elasticity of GK? Why? It turns out that the |C^J|’s for both proteins in glycogen synthesis are greater than one; for GK, much greater than one. Why are very large C^J’s possible in this system? We said a lot about the role of protein kinases in glucose metabolism in liver. What do these results suggest about the ability of protein phosphorylation to regulate the flux in glucose pathways? What is the role of phosphorylation?

Many protein kinases are activated by phosphorylation. How? In some of these protein kinases, the mutation of an activation loop ser or thr to an alanine makes activation impossible. Mutation to a glu or an asp makes activation unnecessary. Explain.

Biochemistry
Exam I
11 February 2004

1. Acetylcholinesterase is inhibited by various organophosphates, including haloxon and nitrophenylhalon:

The rate of loss of activity of the enzyme at a given inhibitor concentration is apparently 1^st order. With NPH, the second inhibitor shown, apparent 1^st order rate constants at 0.3, 0.9 and 3 mM are 0.15, 0.46 and 1.5 per min. What would the 1^st order rate be at 10 mM inhibitor? What kind of inhibition is this? Explain how these compounds inhibit this enzyme. If the enzyme is separated from the inhibitor and incubated in buffer at room temperature, activity returns. This is also an apparently 1 st order reaction. Whether the inactive enzyme was produced with haloxon or NPH, the rate constant for reactivation is 0.017 per min. Suggest an explanation for these observations.

2. Consider the thermodynamics of protein folding. Certain solvents will cause a protein (for example, RNase) to unfold; replacing the solvent with a buffered aqueous solution will cause it to refold. If we think of it as a simple two state system, folded and unfolded protein, what will affect the equilibrium between the two states? Suggest reasons why some solvent changes shift the equilibrium toward the unfolded state, while in water, the equilibrium favors the folded state.

3. We discussed the likely mechanism by which RNase catalyzes RNA hydrolysis. We discussed evidence supporting the catalytic mechanisms of lysozyme and the serine proteases. What kind of evidence would support the catalytic mechanism of RNase that we suggested? Be specific.

4. a. In anemia, the Hb content of the blood is decreased. If we compare a person with 50% of the normal [Hb], to a person poisoned by CO, with 50% of the Hb sites occupied by CO, the anemic individual is much less impaired than the CO intoxicated one. Explain. (CO has 200X more affinity for Hb than O 2.)

b. A mutant Hb has a cysteine replacing Tyr-145 of the b chain. X-ray crystallography indicates that this cys makes a disulfide bond with Cys-93 of the same chain, affecting the position of the C-terminus. How will this affect the following Hb properties? The value of L, oxygen affinity, Bohr effect, Hill constant, BPG affinity.

5. Asp-52 is critical in the functioning of lysozyme. Predict the likely effects of changing this residue to a glutamate; or an alanine. When lysozyme is incubated with (NAG)₄, the slow formation of (NAG)₆ and (NAG)₂ is observed. Explain. How would these mutants be likely to affect this reaction?

6. Two enzymes involved in glycolysis are NAD dependent dehydrogenases: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). Suggest ways to use what you know about alcohol dehydrogenase to determine and verify the stereospecificity of these two enzymes. You have CH₃CD₂OH available to you.

7. In a Dixon plot, 1/v is plotted against [I] at various [S]. How can this be used to determine the K_I?

BIOCHEMISTRY

1. Let us assume that the Dy in actively respiring mitochondria is 140 mV, and that the DpH is 0.4 units. If [P_i] is fixed at 5 mM, and the synthesis of one ATP requires 3 protons flowing into the mitochondrion, what will be the ratio of ATP to ADP at steady state?

The free energy of hydrolysis of a single nucleotide from a nucleic acid is estimated to be about -6 kcal/mol. What is the DG^o' of the polymerization reaction? Would you expect this reaction to be irreversible in the cell, or is there some way to make it irreversible?

4. A one mL preparation of SMPs (sub-mitochondrial particles) has added to it all the remaining Krebs cycle enzymes. Tell what reaction(s) would take place if you added 1 mmol acetyl-CoA. What if you added 1 nmol citrate, besides the acetyl-CoA? Let's add those two plus 1 mmol NAD⁺, and an excess of GDP and P_i. Now what happens? Would there be any additional reaction if you added some DNP? Be as quantitative as possible.

5. Let us assume that you are able to measure both O₂ production and ATP synthesis in intact chloroplasts. If you add DCMU to your chloroplasts and illuminate them, what will you see in your measurements? What if you add DCMU, an electron donor (such as ascorbate) that can reduce plastocyanin, and illuminate? Finally, you add DCPIP (dichlorophenolindophenol), DCMU and illuminate (DCPIP donates electrons to PQ). What do you expect to find in your measurements?

Electron transport can be forced to run in reverse in mitochondria. Can this happen in chloroplasts (or thylakoids)? Why or why not? What would happen in reverse electron transport in thylakoids?

You must answer every question. Please answer each question on a separate sheet of paper and limit your answer to one page (one side only). Be as specific as possible. Confine your remarks to relevant detail. This is an open book exam, so I don't need to know that you can cite the whole TCA cycle or all of glycolysis (for example) - we can take those things for granted.

1. Discuss how the regulation of the Krebs cycle (excluding pyruvate dehydrogenase) illustrates the principles of pathway regulation that we discussed in class.

2. a) A recent food supplement fad has centered on Coenzyme Q (ubiquinone) as a means of increasing a subjective feeling of energy. How might a ubiquinone deficiency or excess affect the Krebs cycle and electron transport in mitochondria?

b) Acetyl-CoA is of central importance in both carbohydrate and fatty acid metabolism. Describe conditions under which you would expect elevated or depressed levels of acetyl-CoA. What effects would such elevation or depression have?

3. a) Arsenate can replace phosphate in many biochemical transformations (for instance, the reaction catalyzed by glyeraldehyde 3-phosphate dehydrogenase). Arsenate esters are unstable and hydrolyze rapidly in water. What effect would the addition of arsenate have on an anaerobic organism doing fermentative glycolysis?

b) Ethanol is metabolized, mostly in the liver, by a series of two NAD-dependent dehydrogenases. The consumption of alcohol after strenuous exercise sometimes leads to dangerous hypoglycemia (low blood sugar). Explain.

4. Discuss the enthalpy and entropy changes associated with adding a small amount of each of the following to water: hexane, acetone, or glucose.

5. a) Draw a graph representing the ratio of O₂ produced to number of incident photons as a function of light intensity for chloroplasts under continuous illumination. Describe as completely as possible what will happen (be sure to include O₂ and ATP production) in a suspension of thylakoid membrane (with a terminal electron acceptor) under continuous illumination and the following conditions: a) no added ADP; b) added ADP and P_i; c) added ADP and P_i, plus an uncoupler such as FCCP.

b) Most of the proton pumping in thylakoids is thought to be coupled to electron transport between the two photosystems. If the DpH is 4, the Dy is 0 and plastoquinone has the same electrode potential as UQ, what would the electrode potential of plastocyanin have to be so that one pair of electrons passing between the photosystems would result in the synthesis of one molecule of ATP? In the above experiments, what would happen if the acceptor took electrons from plastocyanin instead of from photosystem I?

1. It is generally accepted that mammals are unable to convert fats into sugars, and so are unable to survive solely on fat. However, we have seen that some carbon from fat can be converted to sugar, in particular from odd-chain or branched fatty acids (like C₁₇ fatty acid or phytanic acid). Trace this conversion (from fatty acid to sugar) as completely as possible. Indicate which coenzymes are required, and in what steps.

2. The mechanism of the class I aldolases is shown below. Explain the chemistry of this catalysis, paying particular attention to the categories given by the text.

3. a) What would be the effect on glycolysis if PFK-1 were not cooperative? In other words, explain why "normal" Michaelis-Menten kinetics would be an unsatisfactory property for regulatory enzymes.

b) The text derives the higher order kinetics of cooperativity based on a rapid equilibrium model. Describe this model and its assumptions briefly and clearly. Another way to model cooperativity is to assume that substrate binding is unchanged, but that kcat increases in the R state. How would this affect the kinetics? Show the difference between Michaelis-Menten kinetics and this type of cooperativity graphically, and explain.

4. Carnitine deficiency in muscle is manifested in weakness and fatigue, and the accumulation of triacylglycerol droplets in the cytoplasm. In liver, it results in hypoglycemia. Explain.

5. How do the authors of the paper on p53 eliminate the possibility that the exonuclease activity is due to an enzyme that binds tightly to p53, rather than to p53 itself?

6. Consider the paper on protein localization. The data in Table III show proteins that leave the TGN slowly, and others that leave rapidly. What do the authors suggest causes the difference between these two groups? Why do the data for A-ALP and (F/A)A-ALP appear inconsistent with this idea? Explain how these data can be understood to actually be consistent.

7. a) In the paper on CPT induction, what is the purpose of adding cycloheximide in the experiment shown in Fig. 3?

b) Table II shows insulin secretion under different conditions. Discuss the effects of fatty acid and glucose exposure on insulin secretion, and possible inferences from these effects about control of insulin secretion.

And here's one more you can try. Consider it "extra credit" if you want to take a stab at it. If we assume that chloroplasts can synthesize ATP at a maximal rate of 100 nmol/mg chlorophyll per second, and if we assume that we are able to achieve an interior succinate concentration of 10 mM, how much ATP do we expect to make, maximally, per mL of thylakoid lumen? If we assume that each mL of lumen corresponds to 10 mg chlorophyll, how long will this synthesis take?

You must answer all five questions. Please answer each question on a separate sheet of paper and limit your answer to one page (one side only). Be as specific as possible. Confine your remarks to relevant detail. This is an open book exam, so I don't need to know that you can cite the entire purine synthesis pathway (for example) - we can take those things for granted.

1. Consider in vitro fatty acid synthesis under the following conditions: buffered solution containing CoASH, CO₂, citrate, NADPH, ATP, acetyl-CoA carboxylase, fatty acid synthase and citrate lyase. You wish to study this system by labeling the citrate with ¹⁴C and looking for the label in palmitic acid. Can you do so in this system? How many differently labeled citrates can you make (be careful)? As specifically as possible, predict the labeling pattern in the product palmitate for each type of labeled citrate. What would be the labeling pattern if you used¹⁴C-CO₂? Write a balanced equation for the synthesis of palmitate in this system.

2. a) If you could develop an inhibitor for purine nucleoside phosphorylase, what effect would it have on patients with Lesch-Nyhan syndrome? Why is this enzyme not used for purine salvage (rather than hypoxanthine-guanine phosphoribosyltransferase)?

b) Purine nucleotides feedback and inhibit their own synthesis. They also stimulate pyrimidine synthesis (by allosterically affecting the initial enzymes in the pathway). Pyrimidine nucleotides can inhibit their own synthesis, but generally have no effect on purine synthesis. Why should this be?

3. a) The last step in the synthesis of AMP from IMP is catalyzed by adenylosuccinate lyase. Suggest a (step-by-step) mechanism for this reaction. Be sure to include the contribution of the enzyme. Would a coenzyme be required? If so, which? For each step in the sequence, tell which of the five categories of catalysis is involved (see p. 177).

b) Serine can be deaminated to pyruvate (see prob. 11.14b). What coenzyme is required in this reaction? Suggest a mechanism (step-by-step) for this reaction. Include the contribution of the enzyme.

4. a) What assumptions underlie the symmetry model of enzyme cooperativity? Give an example that illustrates the conditions under which these assumptions are valid; include a discussion of how the enzyme structure works to make the assumptions valid. Under what conditions (i.e., what kind of protein structure) would these assumptions not be valid?

b) What is the Hill coefficient? For a cooperative enzyme, is a large or small Hill coefficient better? Why?

5. a) Many, if not most, hormones work through second messenger systems. We talked briefly about a few of them. In general, these second messenger pathways terminate in protein phosphorylation. We know that enzyme activity can be modified by either allosteric effects or by covalent modification. Why should hormone effects be largely the latter? That is, can you suggest the advantages of covalent modification over allostery for hormone signalling?

b) The interaction of light with rod cells is similar to some hormone pathways. How? What is the role of protein phosphorylation in this process? Suggest how you think rhodopsin phosphorylation might be controlled in rod cells.

1. a. Consider the following variants of chymotrypsin, where a single amino acid replacement has occurred affecting the active site residues shown. For each variant, describe the effect you expect on the catalytic activity of chymotrypsin as a result.

If the active site residues have pK_a’s shown, how will catalytic activity vary with pH (assume protonation states of other residues are not important)?

b. Let’s try something similar with pyruvate dehydrogenase (PDH). If the lysine (to which lipoic acid is attached) of dihydrolipoyl transacetylase is changed to an isoleucine, how will the activity of PDH be changed? Will any reaction take place? Give the mechanism of the reaction(s) that you believe will happen.

2. a. Succinyl CoA synthetase is thought to phosphorylate GDP by way of a phosphorylated histidine on the enzyme itself. More than one histidyl-phosphate can be imagined. Draw the structure of the one you think is formed. Nitrogen derivatives of acids (amides) are usually more stable than esters (that is, they are usually not good acyl donors). Why is histidyl-phosphate a good phosphate donor (it must be in order to make GTP from GDP)?

b. Many enzymes that catalyze the isomerization of sugars do so through an enediol intermediate, and general acid and base catalysis. Suggest a mechanism for converting glucose to fructose (or the reverse) that follows this outline.

3. Pyruvate kinase catalyzes the final step in glycolysis. Its activity is affected by many ligands. Some representative data are given below. Velocities are given in mM/min. FBP is fructose 1,6-bisphosphate. Phe is phenylalanine. The PEP concentrations for the phe experiment only are given in the fourth column. At very high [PEP], v_o is 0.13 in the presence of phe.

What kind of effectors are FBP and phe? Plot the data directly as v vs. [S]. What can you tell about the enzyme from these plots, both in the presence and absence of effectors? Can you determine a K_m for PEP under any of these three conditions? Make Hill plots and determine the Hill coefficients. What does this tell you about the enzyme? Do you think these effectors are physiologically relevant? Why or why not?

b. Whole mitochondria will sustain respiration with either succinate or pyruvate, but not with added NADH. SMPs (submitochondrial particles) require succinate, or can oxidize added NADH. Explain. What ratios of succinate/O₂ and pyruvate/O₂ do you expect for these experiments? What other electron donors could be used by either system? If the reaction medium is only weakly buffered, what will happen to the pH in each case? K₃Fe(CN)₆ (ferricyanide) can accept electrons from cytochrome c. If we measure O₂ consumption and ATP production simultaneously, predict the effect(s) (relative to control) of adding ferricyanide, dinitrophenol and oligomycin separately. For instance, if I add ferricyanide to my respiring control, will O₂ consumption and/or ATP production change? How and why? Will the results be different for mitochondria than for SMPs?

1. We addressed in class the effect of alcohol consumption on gluconeogenesis. Consider the effect on other metabolic pathways. Specifically, what would be ethanol's effect on glycolysis and gluconeogenesis in a well-fed person? What effect would it have on fatty acid metabolism (both synthesis and oxidation) and the Krebs cycle? Again, consider both the well-fed and early fasting individual.

2. A baby boy was weaned at 7 months and put on a diet of cow's milk and sugar (sucrose). The baby vomited and refused this food. By trial and error his mother established that sugar and sweetened food made him vomit, and changed his diet to one completely without sucrose. He developed normally after that. A brother was born when the first boy was 9. He was weaned at 5 days and given infant formula with added sugar. He vomited repeatedly, lost weight and became lethargic. On admission to the hospital he was sweating, had convulsions, no reflexes, an enlarged liver and jaundice. Blood sugar, tested by the reducing sugar method, was normal; a glucose oxidase test showed glucose to be very low. The urine also contained reducing sugar, which was identified as fructose by paper chromatography. Withdrawal of sugar (sucrose) from his diet led to rapid improvement. A fructose intolerance test 2 weeks after discharge showed a marked drop in blood glucose and serum phosphate, along with a rise in blood fructose. Blood insulin levels remained constant. Besides what the book tells you about fructose metabolism, you should also know that fructose 1-phosphate inhibits the glycolytic aldolase. Explain the observed effects on the basis of fructose metabolism. What defect did these boys have? Why is glycogen not mobilized under these conditions? Glucagon fails to relieve the hypoglycemia. Why? Most of these clinical signs are not observed in patients with essential fructosuria, a defect in fructokinase. However, blood fructose levels are just as high. Explain. Lastly, suggest a reason for the effect of fructose 1-phosphate on glucokinase.

3. Adipocyte lipolysis is under complex hormonal regulation. We learned that glucagon stimulates lipolysis and insulin inhibits it. Several local signals also influence lipolysis. In particular, adenosine is released by adipose tissue and inhibits lipolysis through a G protein coupled receptor interacting with adenylate cyclase. Nicotinic acid seems to have a similar effect. On the other hand, TNF-b stimulates lipolysis. Consider the data on the back that was collected to try to discover the mechanism of action of TNF-b. Adenosine deaminase will destroy extracellular adenosine. PIA is an adenosine agonist, or analog. Briefly explain how adenosine reduces lipolysis in adipocytes. Explain what each of the figures shows. From these data, explain how TNF-b counteracts that effect. Could this be a general method by which hormones signals can be integrated into a single response? Explain.

4. In one inherited metabolic defect, patients present a complex set of symptoms, including a decreased red blood cell count (and of course, the corresponding decrease in hemoglobin) with the associated anemic symptoms; and the development of fine needle shaped crystals in the urine after standing (about 1.5 g per 24 h). These crystals can be identified as orotic acid. Treatment with uridine alleviates all symptoms. What is the defect? Explain where the symptoms come from. Why does uridine treatment help? Would treatment with cytidine help? Explain. An abundance of PRPP stimulates purine synthesis, leading to gout. Would you expect to see this in these patients? Explain.

All of your answers need to be specific. Avoid vague and general statements. Do not just copy out large portions of the book. Justify your answers with reasons related to the context of the questions.

1. A young man of 15 named Alex K. was taken to the doctor because his mother was concerned about his inability to engage in any strenuous exercise. Alex frequently experiences painful muscle cramps if he attempts anything strenuous. He appeared normal at rest or undergoing light exercise. His liver was normal in size but his muscles were flabby and poorly developed. His blood glucose was normal fasting or fed.

a. After injection of a high dose of glucagon, Alex's blood sugar rose dramatically.

b. Biopsies revealed normal glycogen content in the liver, but elevated content in the muscles. Glycogen structure was normal in both tissues.

c. During ischemic (anoxic) exercise, Alex's blood lactate remains low and steady, but rises quickly to levels 5X higher in normal patients undergoing ischemic exercise (this is just strenuous exercise again). Myoglobin, a muscle protein, was excreted in the urine. Alex's blood alanine level dropped, whereas in normal patients, it goes up somewhat.

d. The physician suggested that Alex avoid strenuous exercise, or consume drinks high in glucose or fructose frequently during exercise.

Alex's symptoms are due to an enzyme deficiency; suggest one or more possibilities (you might wish to know that many of the enzymes of carbohydrate metabolism have different isozymic forms in liver and muscle) and how they might explain these observations.

2. DCMU blocks both ATP synthesis and O₂ evolution in chloroplasts under normal conditions. Addition of an exogenous electron acceptor for PSII restores O₂evolution but not ATP synthesis. Why? When the [NADPH]/[NADP⁺] is high in chloroplasts, PSI donates electrons to quinone rather than to the chain leading to NADPH. Where do the electrons go from here? Is O₂ evolved in this process? Is ATP made? Explain. Will DCMU affect ATP synthesis and/or O₂ evolution when [NADPH]/[NADP⁺] is high? Explain.

3. When O₂ is added to yeast fermenting glucose at a high rate, the rate of glucose consumption declines rapidly and the accumulation of lactate ceases. Explain what causes this change. Give particular attention to the specific enzymes involved and how their activities are modulated.

4. When glucagon increases in the blood, ketone body synthesis starts to rise. Explain how this happens and why (that is, what functions are served by this synthesis). What is the affect on carbohydrate metabolism?

5. The last reaction of the Krebs cycle, catalyzed by malate dehydrogenase, has a D G°’ of +8 kcal/mol. What is the equilibrium constant for the reaction? [malate] is about 0.2 mM and [NAD]/[NADH] is typically 10. What is the concentration of oxaloacetate in the mitochondrion if the pH is 7? If the diameter of the mitochondrion is 2 m m, how many molecules of oxaloacetate are present (assuming a spherical shape)?

6. Much of the fatty acid synthesis in the body occurs in the liver, rather than in the adipose tissue. Under what conditions would you expect fatty acid synthesis to be a major activity in the liver? Be specific and include both external conditions (blood) and internal.

1. There are a large number of glycogen storage diseases. These are diseases that manifest themselves, in part, as an abnormality in the way glycogen is metabolized, especially in muscle. In some of these individuals, the abnormality is manifested as an increase in muscle glycogen, and muscle weakness, an inability to engage in sustained exercise. When insulin levels are low, for instance after fasting, or when epinephrine is administered, these individuals' plasma lactate levels do not rise during maximum exertion, as they do in normal individuals. Even after prolonged fasting, glycogen levels remain high in the muscle. Other tissues (like liver) seem to engage in normal carbohydrate metabolism. There are two enzyme deficiencies that give rise to these symptoms. One is a lack of phosphorylase; the other is a lack of PFK-1. Tell how each of these deficiencies account for the symptoms. How could you distinguish which patients have which deficiency?

Where the following DG's apply: 1. -5 kcal/mol 2. +0.2 kcal/mol 3. -7 kcal/mol 4. -0.5 kcal/mol 5. -6 kcal/mol 6. -5 kcal/mol. Which steps are likely candidates for regulation? Explain. For the transformation, F ® A, which enzymes above could be used? Explain. Which step is the most important step to regulate the level of F? Explain. What substance might be an allosteric ligand of the enzyme that catalyzes this step? Explain. Draw a graph of what you might expect for v vs. [S] for this enzyme. On the same plot, show v vs. [S] in the presence of the above ligand. Explain.

3. a. In many anaerobic organisms, most of the Krebs cycle is still operative. Why? In these organisms, it is usually called the reductive Krebs cycle. Suggest a reason for this term. What part of the cycle could run in reverse? What would be the overall DG°' for this part of the cycle? How could the rest of the cycle intermediates be made?
b. Succinyl-CoA synthase catalyzes the only substrate level phosphorylation in the Krebs cycle. It is known that the enzyme is phosphorylated during catalysis. Suggest a sequence of steps to show how this reaction occurs and how ADP is phophorylated.

4. a. You prepare isolated intact mitochondria. Which of the following substrates can not be used to sustain respiration in this preparation? Why not?

For those that can be used, what else must be supplied for respiration to take place? Why? How much O₂ would be consumed per substrate molecule in each case (assuming no other organic oxidizable substrate is present)? Explain.

b. In the experiment that showed that the Q cycle exists, antimycin, ascorbate, succinate and O₂ are added, in that order. What was observed that supported the Q cycle? What would have been observed if ubiquinol reduced cyt b and the semiquinone reduced cyt c₁ (rather than the other way around)? What if antimycin blocked the oxidation of the semiquinone by cyt b? In each case, what would happen if, finally, ADP and phosphate were added? Would electron transport proceed normally, or would it be different and in what way(s)?

c. Assume that in the mitochondria, [NADH] = 10^-4 M, [NAD⁺] = 10^-3 M, and [O₂] = 10^-4 M. What is the DG (not DG°') for electron transport per pair of electrons? You measure the Dy and find that it is -0.17 volts inside. The pH in the weakly buffered suspension drops to 7.4 and the internal mitochondrial pH is found to be 7.9. How many protons can be pumped per electron passing through electron transport?

2. The legal blood alcohol limit is 0.08%. If this is weight/volume, what is the concentration of ethanol? Ethanol is metabolized mostly by alcohol dehydrogenase and aldehyde dehydrogenase acting in sequence in the liver. The first is a cytoplasmic enzyme, the second is in the mitochondrion. Both enzymes use NAD⁺ and can work with a variety of substrates (methanol, ethanol, etc.). Using the reduction potentials, determine the (DG^o' for each reaction, starting with ethanol. Alcohol oxidation is the rate-limiting step in removing alcohol from the blood. Kinetic data for the liver enzyme is given below. In all cases, [enzyme] = 10^-7 M, and reaction volumes are 3 mL.

Estimate the V_max and the K_m's for both alcohol and NAD⁺. Is the enzyme saturated at the legal alcohol limit? If the liver is the only site of alcohol metabolism, the concentration of alcohol dehydrogenase is 10 mM and the liver volume is 1 L, what is the maximum rate of alcohol metabolism? That is, how much alcohol can be cleared from the body in, say, 1 hour? FYI, alcohol distributes throughout the aqueous volume of the body, which is effectively about 40-50 L.

3. Many tissues and organisms survive on fermentation rather than respiration, at least at some times. If we assume that enzymes exist to phosphorylate most -OH groups, which of the following carbon sources can be fermented, given what we have covered: fructose, glycerol, ethanol, glycerate, and alanine? Outline the pathway that will be followed and the yield of ATP per molecule of carbon source, and give the overall reaction for each material that can be fermented.

4. Consider some experiments with SMPs (submitochondrial particles, ISO vesicles made from fragmented inner mitochondrial membrane). Assume that the reaction volume is always 1 mL, and that O₂ is readily available. Explain your responses.
add 1 mmol pyruvate, what will happen?
Add 1 mmol succinate, what will happen?
Does anything else need to be added in a or b for respiration to take place? If so, what? How much O₂ will be consumed in each case?
Add X (which donates an electron to cyt b_L). Can X sustain oxidative phosphorylation, assuming other needed reagents (except other electron donors) are added? How many protons will be pumped per pair of electrons coming from X?
If you add 10 mmol succinate and 10 mmol NAD⁺, a small amount of NADH is formed. How does this happen? What effect would adding oligomycin have on this result? DNP?

5. a) Draw a graph like those in Fig. 17.16, with the same Y-axis, but with flash duration along the X-axis (up to 10 ms). Explain why the graph looks the way it does.
Suppose that the graph in Fig. 17.24 gave essentially a straight line, rather than the oscillations it does show. What would that mean?
If a suspension of chloroplasts is illuminated with red light (690 ± 10 nm), an oxidation of cytochrome f can be observed, but no O₂ is evolved. Explain. If, in addition, green light (550 ± 10 nm) is used, O₂ evolution is seen. If the amount of glyceraldehyde 3-phosphate produced by the chloroplasts as a function of the ratio of red/green light shows a maximum, with decreasing yields at higher and lower ratios, what would that mean? If the production is maximal at a ratio of 0.25, what would that indicate to you?
What is the cost to the chloroplast of synthesizing the glyceraldehyde 3-phosphate that it exports to the cell, from CO₂? What, then, does this material yield in the cell as it is converted to CO₂? Can you estimate the efficiency of this process? If the chloroplast was organized more like the mitochondrion, and could export ATP directly to the cell, would this be better? Explain.

[PEP] (mM)	V_o	V_o +FBP	[PEP] (mM)	V_o +phe
0.02	0.006	0.09	0.02	0.018
0.03	0.01	0.104	0.033	0.024
0.055	0.031	0.115	0.065	0.032
0.085	0.065	0.118	0.13	0.039
0.15	0.11	0.124	0.325	0.057
0.2	0.125	0.13	3.25	0.094

[NAD⁺] = 0.88 mM		[alcohol] = 7.4 mM
Velocity	[alcohol]	Velocity	[NAD⁺]
8 mM/min	0.5 mM	6.9 mM/min	0.24 mM
6.8	0.14	6.3	0.11
5.6	0.088	5.6	0.067
4.8	0.063	5.0	0.049