FINAL EXAMAll answers are to be written into the Blue Book. Leave the first inside page blank for scoring.METABOLIC BIOCHEMISTRY Winter 2005Immo E. Scheffler FINAL EXAMAll answers are to be written into the Blue Book. Leave the first inside page blank for scoring.There are 16 questions. Make sure that each answer is clearly identified with the question number at the top or left side of the page.Consider the statement on the back of the Blue Book; fill it out and sign it if you want to have your exam returned in the hallway outside of 3234 Bonner Hall. *********************************************************************************QUESTION 1 [12 points]a) The mitochondrial electron transport chain contains four protein complexes and two “mobile carriers”. What are the mobile carriers and in which step(s) are they involved? (A simple schematic diagram with labels is required). [8]The mobile carriers are ubiquinone (coenzyme Q) and cytochrome. Ubiquinone carries electrons from complexes I and II to complex III. Cytochrome c carriers electrons from complex III to complex IV. b) What is a major function of iron-sulfur enters ([Fe-S]) in proteins in the electron transport chain?. [4]A major function of iron-sulfur centers in the subunits of the ETC is to make the proteins capable of conducting electrons. Pure proteins are insulators. Appropriately spaced [Fe-S] centers allow electrons to be passed from one center to the next over a considerable distance (or to ubiquinone).QUESTION 2 [14 points]The following diagram is a schematic representation of the oxygen concentration in a chamber with mitochondria suspended in an appropriate phosphate buffer. Various reagents are added as indicated, with the result that oxygen consumption is either stimulated or arrested. a) The following is a list of substrates, inhibitors, etc., and you are to match them with the compounds A - E shown in the diagram:1) rotenone2) succinate3) hydroxybutyrate (to make NADH)4) ADP 5) antimycinA: ADPB: -hydroxybutyrate (leads to production of NADH and NADH oxidation by complex I)C: rotenone (inhibitor of complex I)D: succinate (is oxidized by complex II)E: antimycin (an inhibitor of complex III)The alternative is to add -hydroxybutyrate first, and then ADPb) Instead of ADP one could have used the uncoupler dinitrophenol. Explain briefly.The ADP is added to provide a substrate for complex V and hence a mechanism for protons to return to the mitochondrial matrix; in the absence of ADP an artificial uncoupler like dinitrophenol can be used to provide an alternate path for the return of the protons pumped by the electrontrnasport chain.QUESTION 3 [12 points]a) In substrate level phosphorylations there is a definite stoichiometry between the amount of inorganicphosphate incorporated into a tri-nucleotide (ATP or GTP) and the amount of substrate (e.g. succinyl-CoA) consumed. In oxidative phosphorylation in mitochondria the amount of NADH oxidized and the amount of ATP produced are not related by an integral number. Explain briefly. [8]An example of substrate level phosphorylation is the following reaction:Succinyl-CoA + Pi + GDP succinate + GTP + CoASH (a reaction in the Krebs cycle)In this case there is an obligatory one-to-one relationship between the amount of succinate produced and the amount of ATP made (or Pi consumed, etc.). the reaction is carried out in the active site of a single enzyme where all the reactants bind .In oxidative phosphorylation in mitochondria there are two major components. The first is the electron transport chain; substrates like NADH and succinate are oxidized and electrons are transferred through a series of complexes to oxygen. As electrons pass through complexes I, III, and IV, protons are pumped across the inner membrane from the matrix to the intermembrane space. This sets up an electrochemical gradient consisting of a proton gradient (chemical), and a membrane potential (because the proton carries a positive charge). The electrochemical gradient represents a form of stored energy derived from the oxidation of NADH (or succinate). A physically distinct complex (ATPsynthase, or complex V) at a separate location in the inner membrane can exploit the electrochemical gradient to carry out the endergonic ATP synthesis. This is achieved by a mechanism in which protons returning to the matrix through complex V drive a rotor within the complex. The rotation forces the active site for ATP synthesis through an enzyme cycle converting ADP and Pi into ATP.The number of protons pumped by the ETC determines the maximum number of ATPs that can be synthesized per NADH oxidized, but there are numerous other mechanisms for protons to return to the matrix. In the extreme case (artificial uncoupler or natural uncoupling proteins present) the proton circuit can operate without the synthesis of ATP. b) Thermogenesis in brown adipose tissue is achieved by means of an uncoupling protein. What, precisely, is the function and role of this protein? What would be the P/O ratio when a large excess of uncoupling protein is present?[4]See above: when uncoupling protein is present in excess (as in brown adipose tissue), all the protons pumped by the ETC return to the matrix through the UCP; no ATP is produced, and theP/O ratio approaches zero. All the free energy released from electron transport (oxidations) is dissipated as heat.QUESTION 4 [18 points]In -oxidation of a saturated C18 fatty acid (stearic acid) a reaction sequence is repeated over several cycles.a) Illustrate one such cycle with structural formulae [8]The above scheme illustrates one cycle starting with palmitic acid (C16). The formulae should clearly show the carboxyl group in thioester linkage to CoA, and the changes involving the alpha and beta carbons. The explicit names of the enzymes are not required.b) How many cycles are required to convert this acid completely into acetyl-CoA? [2]For stearic acid (C18) one would require 8 cyclesc) How many NADH are produced after the fatty acid has been completely converted to acetyl-CoA? [2]Each cycle produces one NADH, i.e. a total of 8 NADH would be produced d) What is the other co-factor reduced in this process? It is not free in the matrix and is immediately re-oxidized with the electrons transferred to what? [2] The other co-factor is FAD which is associated with the enzyme carrying out the first oxidation togenerate the double bond; FADH2 is produced but
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