MIC328_ProblemSet1

Problem Set 1

1. To convert glucose to glucose 6-phosphate or fructose 6-phosphate to fructose 1,6 biphosphate, the catalyzing enzyme must mediate two reactions, one with a -∆G and one with a +∆G. Using structural formulae, diagram the two reactions that comprise the conversion of glucose to glucose 6-phosphate, and the conversion of fructose 6-phosphate to fructose 1,6 biphosphate. Indicate the ∆G of each reaction and identify the enzyme that catalyzes each of these steps in glycolysis.

2. Fructose 1,6 biphosphate is cleaved during glycolysis yielding two 3 carbon molecules. What are the structural formulae and names of these molecules? What enzyme catalyses the cleavage of fructose 1,6 biphosphate? Which of the two molecules is directly on the glycolytic pathway? What must occur to the other molecule before it can continue in the glycolytic pathway?

3. Using structural formulae diagram the reactions catalyzed by the following enzymes:

    A. Glyceraldehyde 3-phosphate dehydrogenase.
    B. Phosphoglycerate kinase.
    C. Phosphoglycerate mutase.
    D. Enolase.
    E. Pyruvate kinase.
    F. Triose phosphate isomerase.

4. High levels of ATP can lead to elevated levels of glucose or glucose 6-phosphate, but not fructose 6-phosphate
    A. What enzyme is inhibited by high intracellular ATP concentrations?
    B. Why did evolution favor ATP as the inhibitor?
    C. Why does glucose or glucose 6-phosphate accumulate?
    D. What enzyme is inhibited by glucose 6-phosphate and what reaction does it catalyze?
    E. What two properties are associated with the committed step of a pathway? Hexokinase does not catalyze the committed step of glycolysis, why?

5. In two glycolytic steps, ATP is synthesized via substrate level phosphorylation. Using structural formulae, diagram these steps. Remember that each of these steps is the summation of two subreactions, one with a -∆G and one with a +∆G. Indicate the ∆G of each subreaction. What must be true about the energy released by hydrolyzing the phosphate bond in the glycolytic intermediate to support the generation of ATP?

6. Most bacteria utilize the pentose phosphate and glycolytic pathways concurrently. What are the primary benefits of the pentose phosphate pathway?

7. The pentose phosphate pathway converts glucose to the 5-carbon sugar ribulose by oxidizing glucose.

A. Using structural formulae draw these reactions.
B. What important co-enzyme is regenerated by these reactions, and for what will it be utilized?

8. Like glycolysis, the Entner-Doudoroff pathway converts glucose to two molecules of pyruvate. However, the Entner-Doudoroff does not contain a fructose intermediate. Using structural formulae, diagram this pathway. What important co-enzyme is generated in the Entner-Doudoroff pathway that is NOT generated in glycolysis? How is this co-enzyme generated in organisms that do not contain the Entner-Doudoroff pathway?

9. The ability to make partial diploid bacterial cells for the lac operon was crucial in determining the roles of the individual trans and cis-acting elements. For the examples of haploid and diploid cells given below, indicate whether the transcription of the lacZ genes would be inducible, non-inducible or constitutive and whether the cell could grow with lactose as its only carbon source.

Nomenclature:
    o⁺   wild type operator.
    o^c   an operator that cannot bind the repressor.
    i⁺   wild-type repressor.
    i^-    repressor that cannot bind the operator.
    i^d   repressor that cannot bind lactose.
    z⁺ wild-type beta-galactosidase protein.
    z^-    beta-galactosidase protein with no enzymatic activity.

Haploid cells

i⁺o^cz^-                i^do^cz⁺            i^-o⁺z-            i^do⁺z⁺

Partial diploid cells

i^do⁺z⁺/i⁺o^cz^-             i^-o⁺z⁺/i⁺o⁺z^-        i^do^cz⁺/i^-o⁺z^-        i⁺o⁺z-/i^-o⁺z⁺

10. Describe the relative level of lac operon transcription (high, low, none) and the level of cAMP (high, low) that would be found in cells utilizing the following carbon sources.

Carbon source	lac operon transcription	cAMP levels
glucose
maltose
lactose
glucose and lactose
glucose and maltose
lactose and maltose

11. How does the level of environmental glucose regulate intracellular cAMP levels?

12. What enzyme converts oxaloacetate and acetyl-CoA to citrate? Using structural formulae draw the reaction. Why is this the committed step of the TCA cycle, and what molecules inhibit enzymatic activity?

13. One enzyme in the TCA cycle carries out a two-step reaction, which goes through an intermediate. What is the name of this enzyme? Using structural formulae draw the reactions.

14. The oxidation of isocitrate, an alpha hydroxy acid, into oxalosuccinate, an alpha keto acid, requires a coenzyme, whereas the spontaneous decarboxylation of oxalosuccinate does not require a co-enzyme. Explain.

15. What types of reactions do dehydrogenases catalyze? What types of co-enzymes do they usually require? What four reactions in the TCA cycle utilize dehydrogenases. Using structural formulae diagram these reactions.

16. The reaction catalyzed by succinyl CoA ligase generates succinate and GTP. This reaction is the summation of two subreactions, one with a -∆G and one with a +∆G. Indicate the ∆G of each subreaction. Using structural formulae, diagram the conversion. What must be true about the energy released by hydrolyzing the thioester succinly CoA to support the generation of GTP?

17. What critical difference between the TCA and glyoxylate cycles allows microbes to grow with acetate as their only carbon source? Remember the carbon source must be sufficient for both catabolism and anabolism.

18. What reactions do the TCA and glyoxylate cycles have in common? What two reactions are unique to the glyoxylate cycle? Using structural formula diagram these reactions.

19. Fatty acids can be used as carbon sources for the generation of the energy. Two-carbon units are removed from fatty acids by "oxidize-hydrolyze-oxidize." Remove a two carbon unit from a fatty acid (do not forget the "SCoA" step).

20. Per carbons oxidized to CO2, what molecule is a more efficient for energy storage, glucose or a 6 carbon fatty acid? Explain.

21. Evolution is fond of redundancy.

        A. How is the conversion of succinate to oxaloacetate similar to the oxidation of fatty acids?
        B. What TCA reaction is mechanistically similar to pyruvate dehydrogenase?
        C. What TCA reaction is mechanistically similar to malate synthase?

22. Oxidative phosphorylation can be described as sets of energetically coupled exergonic and endergonic reactions or processes. For the following reactions or processes, indicate whether they are exergonic or endergonic and indentify the process or reaction to which it is energetically coupled.

        A. The reduction of NAD+ or FAD.
        B. The generation of the proton motive force.
        C. The movement of H+ into the cell.

23. Under both aerobic and anaerobic conditions, the cell must regenerate its supply NAD+. How is this done under aerobic conditions? Anaerobic conditions?

24. Under anaerobic conditions, what are the two most common fates of the molecule pyruvate in microorganisms? Using structural formulae draw the reactions in these two pathways?

25. The enzyme pyruvate dehydrogenase requires a co-enzyme, whereas the enzyme pyruvate decarboxylase does not. Explain.