Final Exam Review BiCh

Transcription

1 Final Exam Review BiCh

2 Exam will be posted to the course website on Monday morning, December 4th (not on Wednesday, December 6th as stated in the syllabus) Exams are due by 5pm on Friday, December 8th Exams should be turned in (pick one): Exam Details by , cc ing Belinda, Nadia and Heather as a physical copy to the BiCh110 boxes on the first floor of Church, under the bridge between Church and Crellin across from Schlinger, under the disapproving gaze of AO Beckmann Exam will focus on the second (post-midterm) part of the quarter, but some questions will require the general biochemistry knowledge from the first half of the term

3 Open book, 8 questions, 4 hours (200 points) Resources you may use are limited to: your Stryer Biochemistry textbook, your lecture notes, problem sets, and any material that has been posted on the course website. You may also use a calculator. During this portion of the exam, you may have one break for any amount of time. During the break you may not consult any prohibited resources for exam material and you may not talk to other students about the exam. Exam Details If you run out of time, continue to finish this section but clearly note what work was done past the time limit. You will receive half credit for this work. Unless specifically noted, limit your answers to 5 sentences. At all times use your own words even when you directly refer to book for an answer. If your answer contains incorrect information along with correct information, points will be deducted. Please write your answers to each question on a separate page(s) with your name on the top of every page. (This means no blue books, please)

4 Section 2: Final course survey, 5 questions, unlimited time We want to hear about your experiences with the course and would appreciate your taking the time to answer these survey questions. Exam Details Questions will focus on lectures Also, please fill out the TQFR!! To protect anonymity, there will be a separate pile to turn in these surveys, and names are not required. Please take the survey after you have completed the entire exam.

5 Questions?

6 Practice Problems

7 Given the system below, answer the following with true/false. An enzyme catalyzing the reaction will: Enzyme 1 (a) Increase K eq (b) Increase k1 (c) Decrease ΔG (d) Decrease ΔG 0

8 Given the system below, answer the following with true/false. An enzyme catalyzing the reaction will: Enzyme 1 answer (a) Increase K eq (b) Increase k1 (c) Decrease ΔG (d) Decrease ΔG 0 F T T F

9 Experimental condition KM Enzyme 2 (a) (b) (c) (d) (e) Twice as much enzyme is used Half as much enzyme is used A competitive inhibitor is present An uncompetitive inhibitor is present A non-competitive inhibitor is present Vmax

10 Experimental condition KM Enzyme 2 answer (a) (b) (c) (d) (e) Twice as much enzyme is used Half as much enzyme is used A competitive inhibitor is present An uncompetitive inhibitor is present A non-competitive inhibitor is present Vmax doubles -halves --increase decrease decrease decrease --

11 Enzyme 3 Tryptophan synthase is an enzyme that contains a pyridoxyl phosphate (PLP) cofactor and catalyzes the synthesis of L-tryptophan from L-serine and indole. Addition of L-serine to the enzyme causes an increase in fluorescence (magenta). Subsequent addition of indole reduces the fluorescence (blue). What can be concluded from the fluorescence data? Note: neither free serine nor indole fluoresce at these wavelengths

12 Enzyme 3 answer The fluorescence experiment shows that there is an enzyme-serine complex and an enzyme-serine-indole complex. Bonus: Trp synthase is a cooperative enzyme. What would a titration of Ser in the presence of excess indole look like? How would you change the shape of that titration?

13 Enzyme 3 answer Bonus: Trp synthase is a cooperative enzyme. What would a titration of Ser in the presence of excess indole look like? How would you change the shape of that titration? The titration curve would look sigmoidal. In order to change the shape, we could abolish subunit interactions, or find T and R state affectors.

14 Enzyme 3 answer Bonus: Trp synthase is a cooperative enzyme. What would a titration of Ser in the presence of excess indole look like? How would you change the shape of that titration? The titration curve would look sigmoidal. In order to change the shape, we could abolish subunit interactions, or find T and R state affectors.

15 Metabolism-I 1 Why the regulation of phosphofructokinase by energy charge not as important in the liver as it is in muscle? What are other ways of regulation of the enzyme?

16 Why the regulation of phosphofructokinase by energy charge not as important in the liver as it is in muscle? What are other ways of regulation of the enzyme? Metabolism-I 1 The energy needs of a muscle cell vary widely, from rest to intense exercise. Consequently, the regulation of phosphofructokinase by energy charge is vital. In other tissues, such as the liver, ATP concentration is less likely to fluctuate and will not be a key regulator of phosphofructokinase. It can also be regulated by expression level

17 Metabolism-I 2 Sucrose is commonly used to preserve fruits. Why is glucose not suitable for preserving food?

18 Metabolism-I 2 Sucrose is commonly used to preserve fruits. Why is glucose not suitable for preserving food? Glucose is reactive because its open-chain form contains an aldehyde group

19 Metabolism-I 3 Xylose has the same structure as that of glucose except that it has a hydrogen atom at G-6 in place of a hydroxymethyl group. The rate of ATP hydrolysis by hexokinase is markedly enhanced by the addition of xylase. Why?

20 Metabolism-I 3 Xylose has the same structure as that of glucose except that it has a hydrogen atom at G-6 in place of a hydroxymethyl group. The rate of ATP hydrolysis by hexokinase is markedly enhanced by the addition of xylase. Why? Hexokinase has a low ATPase activity in the absence of a sugar because it is in a catalytically inactive conformation.

21 Redox reactions

22 1. A positive redox potential means a substance has: a. a lower affinity for an electron c. a lower affinity for a proton b. a higher affinity for an electron d. a higher affinity for a proton Practice problem 2.Balance the redox reaction below: MnO4- + I- I2 + Mn2+

23 1. A positive redox potential means a substance has: a. a lower affinity for an electron c. a lower affinity for a proton b. a higher affinity for an electron d. a higher affinity for a proton Practice problem 2.Balance the redox reaction below: MnO4- + I- I2 + Mn2+ Separate the two half reactions: I- I 2 MnO4- Mn2+ Add water to balance O atoms and hydrogen to balance H atoms: 2 I- I2 MnO H+ Mn H2O Balance the electrons: 5(2I- I2 +2e-) 2(5e- + 8H+ + MnO4- Mn2+ + 4H2O) Add them together and cancel redundancies 10 I H+ + 2 MnO4-5 I2 + 2 Mn H2O

24 1. For free elements the oxidation state is zero. e.g. Fe(s), O2(g), O3(g), H2(g), Hg(l) Oxidation numbers 2. For monoatomic ions, the oxidation state is given by the charge on the ion. e.g. Cl- (-1), Fe2+ (+2), Fe3+ (+3), S2- (-2), Ca2+ (+2), H+ (+1) 3. Elements in compounds have common oxidation states. a) hydrogen is +1 (except in metal hydride compounds such as LiH) b) oxygen is -2 (except in peroxides such as H2O2) c) N-R3 is -3 while RN=NR is The sum of the oxidation states is equal to a molecule s charge.

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26 Reduction: 2 electrons gained by carbon Reduction: 1 electrons gained by carbon Oxidation: 4 electrons lost by carbon 0 electrons lost or gained by carbon

27 1) Ligation reactions: form bonds by using free energy from ATP cleavage; necessary for anabolism. 2) Isomerization reactions: rearrange atoms necessary for subsequent reactions such as oxidation-reduction. Other types of reactions in metabolism: 3) Group transfer reactions: activate compounds for subsequent reactions or modify compounds for regulatory purposes (trap glucose in the cell for further catabolism) 4) Hydrolytic reactions: Hydrolysis for the degradation of large molecules for metabolism or to reuse some of the components for biosynthetic purposes 5) Carbon-carbon bond cleavage: release of CO2 or H2O during metabolic pathways that help drive reactions forward.

28 1) Control the amounts of enzymes: Occurs at the level of transcription Three ways cells control metabolism 2) Control catalytic activity: Allosteric control; feedback loops; covalent modifications; energy levels of the cell 3) Control the accessibility of substrates: Compartmentalization of substrates; substrate sequestration

29 Back to pyruvate.

30 Entry to the citric acid cycle through Acytl-CoA

31 Citric acid cycle basics

32 Pyruvate dehydrogenase is highly regulated by acetyl CoA, NADH, ATP, and phosphorylation because it catalyzes an irreversible reaction. Mechanisms and regulation Isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are highly regulated by ADP/ATP, NAD+/NADH, their reactants/products, and up/downstream matabolites because these are the first steps in the cycle that generate electrons (NADH). The regulation of these steps can also shuttle metabolites to other pathways (ex. amino acid synthesis) when energy is in excess.

33 Fluoroacetate is found in certain plants of Autrailia and Brazil and is highly toxic to cattle, other animals, and insects. It is metabolized into fluoroacetyl-coa by the enxyme acetate thiokinase and then is incorporated into fluorcitrate as shown above. A scientist studying the effect of fluoracetate on mammalian muscle cells found a decrease in the rate of glycolysis and in the amount of citric acid cycle intermediates. However, citrate levels increased several fold. Metabolism-II Citric acid cycle a) What step in the citric acid cycle appears to be blocked? Why do citrate levels increase dramatically, while other intermediates decrease? b) Draw the mechanism for the formation of the intermediate at right resulting from incubation of FCH2C*O2Na with muscle cells (*=C14 label). How would this intermediate lead to the three effects of fluoroacetate poisoning described above?

34 Fluoroacetate is found in certain plants of Autrailia and Brazil and is highly toxic to cattle, other animals, and insects. It is metabolized into fluoroacetyl-coa by the enxyme acetate thiokinase and then is incorporated into fluorcitrate as shown above. A scientist studying the effect of fluoracetate on mammalian muscle cells found a decrease in the rate of glycolysis and in the amount of citric acid cycle intermediates. However, citrate levels increased several fold. Metabolism-II Citric acid cycle answer a) What step in the citric acid cycle appears to be blocked? Aconotase Why do citrate levels increase dramatically, while other intermediates decrease? Because of a roadblock b) Draw the mechanism for the formation of the intermediate at right resulting from incubation of FCH2C*O2Na with muscle cells (*=C14 label). How would this intermediate lead to the three effects of fluoroacetate poisoning described above? Citric acid levels increase and inhibit PFK (the commitment step in glycolysis) * * *

35 You should know: Cellular location of steps Reactants (ex. NAD+) and products (ex. ATP, CO2) for each step and the net reaction for the pathway General mechanism for each step (ex. oxidation-reduction reaction vs. isomerization vs. etc) Which enzymes are regulated and how The Citric Acid Cycle You should be able to: Link the citric acid cycle to other pathways discussed in class Discuss how various conditions (ex. high ATP, low O2) would affect the citric acid cycle Follow a carbon atom through the reactions of the citric acid cycle and related pathways Explain why certain enzymes are regulated Count ATP/NADH consumed/generated when various reactants/intermediates are entered into a pathway Explain why a reaction is favorable/unfavorable (ex. why phosphoryl transfer has ΔG) Apply your knowledge of the pathways we ve studied to analyze a new pathway

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37 Complex I Electron transport chain concise summary NADH + H+ + FMN NAD+ + FMNH2 FMNH2 + 2 Fe-Soxidized FMN + 2 Fe-Sreduced + 2 H+ 2 Fe-Sreduced + Q + 2 H+ 2 Fe-Soxidized + QH2 Complex II Succinate + FAD fumarate + FADH2 FADH2 + Fe-Soxidized FAD + Fe-Sreduced Fe-Sreduced + Q + 2 H+ Fe-Soxidized + QH2 Complex III QH2 + 2 cytochrome c (with Fe3+) Q + 2 cytochrome c (with Fe2+) + 2 H+ Complex IV 4 cytochrome c (with Fe2+) + 4 H+ + O2 4 cytochrome c (with Fe3+) + 2 H2O

38 1. The major metabolic consequence of perturbation of electron transfer in mitochondria is which of the following? a. Increased production of NADPH c. Increased reduction of O2 to H2O b. Increased oxidation of NADH d. Decreased regeneration of NAD+ Practice problem 2. A patient has been exposed to a toxic compound that increases the permeability of mitochondrial membranes to protons. Which of the following metabolic changes would be expected in this patient? a. Increased ATP levels c. Increased ATP synthase activity b. Increased oxygen utilization d. Decreased Pyruvate DH activity 3. Which of the following best explains why cytosolic NADH can yield potentially less ATP than mitochondrial NADH? a. Cytosolic NADH loses energy when transferring electrons b. Once NADH enters the matrix from the cytosol, it becomes FADH2 c. Electron transfer from the cytosol to matrix can take more than one pathway d. There is an energy cost for bringing cytosolic NADH into the matrix

39 1. The major metabolic consequence of perturbation of electron transfer in mitochondria is which of the following? a. Increased production of NADPH c. Increased reduction of O2 to H2O b. Increased oxidation of NADH d. Decreased regeneration of NAD+ Practice problem 2. A patient has been exposed to a toxic compound that increases the permeability of mitochondrial membranes to protons. Which of the following metabolic changes would be expected in this patient? a. Increased ATP levels c. Increased ATP synthase activity b. Increased oxygen utilization d. Decreased Pyruvate DH activity 3. Which of the following best explains why cytosolic NADH can yield potentially less ATP than mitochondrial NADH? a. Cytosolic NADH loses energy when transferring electrons b. Once NADH enters the matrix from the cytosol, it becomes FADH2 c. Electron transfer from the cytosol to matrix can take more than one pathway d. There is an energy cost for bringing cytosolic NADH into the matrix

40 ATP Synthase ATP synthase has two subunits (F0 and F1, the channel and the rotor respectively) The F1 subunit has three states: loose conformation: ADP and Pi bind reversibly tight conformation: formation of ATP open conformation: release of ATP It takes about 3.3 protons to form 1 ATP.

41 1. The conduction of protons by the F0 unit of ATP synthase is blocked by dicyclohexylcarbodimide, which reacts readily with carboxyl groups. What are the most likely targets of action of this reagent? a. Arginine and Lysine c. Alanine and Glutamine b. Aspartate and Glutamate d. Glutamine and Asparagine Practice problem 2. Each 360 rotation of the γ subunit leads to the synthesis and release of three molecules of ATP. How many protons are required to form one ATP if the c ring has 12 subunits? What about 14 c subunits? a. 9.4 for 12 subunits; 8 for 14 subunits c. 8 for 12 subunits; 9.4 for 14 subunits b. 4.7 for 12 subunits; 4 for 14 subunits d. 4 for 12 subunits; 4.7 for 14 subunits 3. Why do isolated F1 units of ATP synthase catalyze ATP hydrolysis? a. They don t c. Because hydrolysis of ATP is endergonic b. Because hydrolysis of ATP is exergonic d. Because there is no proton gradient

42 1. The conduction of protons by the F0 unit of ATP synthase is blocked by dicyclohexylcarbodimide, which reacts readily with carboxyl groups. What are the most likely targets of action of this reagent? a. Arginine and Lysine c. Alanine and Glutamine b. Aspartate and Glutamate d. Glutamine and Asparagine Practice problem 2. Each 360 rotation of the γ subunit leads to the synthesis and release of three molecules of ATP. How many protons are required to form one ATP if the c ring has 12 subunits? What about 14 c subunits? a. 9.4 for 12 subunits; 8 for 14 subunits c. 8 for 12 subunits; 9.4 for 14 subunits b. 4.7 for 12 subunits; 4 for 14 subunits d. 4 for 12 subunits; 4.7 for 14 subunits 3. Why do isolated F1 units of ATP synthase catalyze ATP hydrolysis? a. They don t c. Because hydrolysis of ATP is endergonic b. Because hydrolysis of ATP is exergonic d. Because there is no proton gradient The direction of a reaction is determined by the ΔG difference between substrate and products. An enzyme speeds up both the forward and backward reactions. Hydrolysis of ATP is exergonic so ATP synthase will enhance the hydrolytic reaction.

43 What you should know: where the process takes place in a cell which reactants contribute to the electron transport chain and how the flow of electrons is mediated (reduction potentials) Oxidative Phosphorylati on which proteins are involved and how they contribute What you should be able to do: understand how this process relates to the other metabolic processes track the flow of electrons explain the mechanism of ATP synthase explain what factors change its effectiveness

44 Light reactions of photosynthesi s basics

45 Mechanisms and regulation In your homework I altered the size of the Antenna complex. What else would be interesting to change?

46 1. Carotenoids are accessory pigments that can transfer the energy from other wavelengths of light to chlorophyll but cannot release electrons themselves. Considering they are all conjugated polynes, why might this be? (pick two) a. they are too far from the reaction center c. they are not stabilized by enough ring structures b. they don t have as long of tails as chlorophyll d. they are not stabilized by a metal ion Practice problem 2. Which of the following is not true? a. The light reactions of cyclic photophosphorylation involve redox reactions b. The light reactions of cyclic photophosphorylation generate ATP c. The light reactions of cyclic photophosphorylation use only photosystem I d. The light reactions of cyclic photophosphorylation use chemiosmosis e. The light reactions of cyclic photophosphorylation produce NADPH

47 1. Carotenoids are accessory pigments that can transfer the energy from other wavelengths of light to chlorophyll but cannot release electrons themselves. Considering they are all conjugated polynes, why might this be? (pick two) a. they are too far from the reaction center c. they are not stabilized by enough ring structures b. they don t have as long of tails as chlorophyll d. they are not stabilized by a metal ion Practice problem 2. Which of the following is not true? a. The light reactions of cyclic photophosphorylation involve redox reactions b. The light reactions of cyclic photophosphorylation generate ATP c. The light reactions of cyclic photophosphorylation use only photosystem I d. The light reactions of cyclic photophosphorylation use chemiosmosis e. The light reactions of cyclic photophosphorylation produce NADPH

48 Practice problem 3. Pheophytin is similar in structure and function to bacteriopheophytin seen below. What is the most important difference are there between bacterial photosynthesis and plant photosynthesis? a. the direction of the proton gradient b. the presence of the WOC c. the presence of Qb d. the presence of Qa 4. In which way is cytochrome bf and complex III are similar? a. they transfer to a carrier with a heme group b. they transfer 2 protons across the membrane c. they collect 2 protons from the matrix d. they require more than two steps

49 Practice problem 3. Pheophytin is similar in structure and function to bacteriopheophytin seen below. What is the most important difference are there between bacterial photosynthesis and plant photosynthesis? a. the direction of the proton gradient b. the presence of the WOC c. the presence of Qb d. the presence of Qa 4. In which way is cytochrome bf and complex III are similar? a. they transfer to a carrier with a heme group b. they transfer 2 protons across the membrane c. they collect 2 protons from the matrix d. they require more than two steps

50 You should know: Cellular location of the steps Reactants and products for each step and the net reaction for the pathway General mechanisms for each step (ie oxidation-reduction reaction vs isomerization) Which enzymes are regulated and how Photosynthesis You should be able to: Link photosynthesis to other pathways discussed in class Draw analogous pathways Discuss how various conditions (ex ) would affect photosynthesis and how these constraints have led to the evolution of different types of photosynthesis Explain why certain enzymes are regulated Count ATP and FADH2 produced Explain why a reaction is favorable or unfavorable Be able to use these skills to analyze a new pathway

51 Fatty acids have four major physiological roles: 1) Fatty acids are fuel molecules During rest, fatty acids are the primary source of energy Fatty acid metabolism 2) Fatty acids are building blocks of phospholipids and glycolipids Components of biological membranes 3) Proteins are modified by the covalent attachment of fatty acids, which targets the proteins to membrane locations 4) Fatty acid derivatives serve as hormones and intracellular messengers

52 Degradation of Fatty Acids

53 Entry to the citric acid cycle through Acytl-CoA FAD rather than NAD+ is the electron acceptor because G for this reaction is insufficient to drive the reduction of NAD+

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57 1. What is the ATP yield for the complete oxidation of C17 fatty acid? Assume that the propionyl CoA ultimately yields oxaloacetate in the citric acid cycle. Practice problem 1 a. 119 b. 120 c. 118 d. 121

58 1. What is the ATP yield for the complete oxidation of C17 fatty acid? Assume that the propionyl CoA ultimately yields oxaloacetate in the citric acid cycle. Practice problem 1 a. 119 b. 120 c. 118 d. 121 Activation fee to form acyl CoA: -2 ATP 7 acetyl CoA at 10 ATP/acetyl CoA: +70 ATP 7 NADH at 2.5 ATP/NADH: ATP 7 FADH2 at 1.5 ATP/FADH2: ATP Propionyl CoA conversion to succinyl CoA: -1 ATP Succinyl CoA succinate: +1 ATP Succinate fumarate + FADH2: +1.5 ATP Fumarate malate Malate oxaloacetate + NADH: +2.5 ATP Total: 120 ATP

59 Practice problem 2 2) Explain how each of the following dysfunctional mutants (independently) affect fatty acid metabolism: i) Dysfunctional isocitrate dehydrogenase ii) Dysfunctional pyruvate kinase

60 2) Explain how each of the following dysfunctional mutants (independently) affect fatty acid metabolism: i) Dysfunctional isocitrate dehydrogenase Practice problem 2 Isocitrate dehydrogenase is involved in the conversion of isocitrate to α-ketoglutarate with concomitant formation of NADH. As this disrupts the citric acid cycle, citrate is not synthesized, which is necessary for fatty acid synthesis. Thus the mutant removes citrate necessary for fatty acid synthesis. ii) Dysfunctional pyruvate kinase Pyruvate kinase catalyzes the final step in glycolysis to form pyruvate which gets transported into the mitochondrion for the citric acid cycle. Thus the mutant removes pyruvate necessary for fatty acid synthesis.

61 Fatty Acid metabolism You should know: Cellular location of the steps Reactants and products for each step and the net reaction for the pathway General mechanisms for each step (ie oxidation-reduction, cleavage, condensation vs hydration) Which enzymes are regulated and how (ex: commitment to synthesis) You should be able to: Compare synthesis and degradation Draw pathway mechanisms Explain why certain enzymes are regulated Count ATP generated from even, odd or unsaturated fatty acids Track labeled carbons from a fatty acid Explain why a reaction is favorable or unfavorable Be able to use these skills to analyze a new pathway

62 Additional Questions? Questions?