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1 Fa15 BIBC102 final, page 1! Good Evening Done-tabolites! This is your final. The graded exams will be available for pickup sometime next week. Stay tuned. The key will be posted on the website TONIGHT. Feel free to register comments, complaints, questions, or corrections to me by . We will consider those suggestions or observations during the grading process. So thanks in advance to your vigilance I have enjoyed teaching this class. I always learn a lot, and appreciate the energy and dedication you all show. To commemorate the event, and to replenish your glycogen stores, will have a bunch of pizza outside the test room, which we must eat. We have learned to stage the pizze in two waves: those who finish in the First Burst, and those who stay till near the Bitter End. Both strategies work for different people. Happy Holidays! Summation Page 2 (out of 12) Page 3 (out of 10) Page 4 (out of 14) Page 5 (out of 12) Page 6 (out of 12) Page 7 (out of 9) Page 8 (out of 17) Page 9 (out of 16) Page 10 (out of 13) Page 11 (out of 8) Page 12 (out of 13) Page 13 (out of 14) Total (out of 150)

2 Fa15 BIBC102 final, page 2 1) (5pts) I-am-mean-o acids a) The peptide bond is a central feature of all proteins. Using R and R for the R groups, draw two generic amino acids connected by a peptide bond. Clearly indicate the bond. b) Shown over yonder on the right are pictures of R groups found in 6 of the 20 protein structural amino acids. Using the spaces provided, write the 3 letter name for each, and the 1 letter code for each. 2) (4 pts) A site for sore I s The picture shows a schematic arrangement of amino acid R groups arranged into a famous catalytic site. We have discussed this arrangement in class. It allows very effective catalysis by the enzyme called chymotrypsin. Answer the following questions about this arrangement of amino acid R groups that we studied in detail in class a) What is the name of this structural arrangement? b) What are the three and one-letter codes for each? and and and c) What kind of reaction does this arrangement catalyze? One sentence d) Briefly describe the role of each R group in the catalyzed reaction. State the amino acid name (or letter code), and then describe with a brief phrase. List them from left to right in the diagram. 1): 2): 3): 3) (3 pts) It s Ammazing! Glutamate is the cognate amino acid of α-keto-glutarate, which you can draw with Only Marginal Struggle and Grief. The enzyme glutamate dehydrogenase converts one into the other. a) Write the reaction. Include the structure of the amino acid and the α-keto acid, and appropriate abbreviations of any other substrates or products. b) How are this enzyme and glutamate important in amino acid catabolism? One sentence

3 Fa15 BIBC102 final, page 3 4) (10pts) All s well that Enz well- A laboratory is studying an enzyme inhibitor called I. I inhibits its target enzyme by raising the Km without affecting the Vmax. Answer the following questions based on this observation a) Does the presence of I alter the concentration of substrate that is needed to attain 50% saturation of its target enzyme? Yes or no, and why (one sentence) b) Does the presence of I alter the measured kcat of the enzyme? Yes or no, and why (one sentence) c) In the left box provided below, sketch a typical substrate vs Vo plot, showing the behavior of the enzyme in the presence and absence of I. Label both axes, show which curve is measured in the presence and absence of I, include the position of the Km and Vmax for each curve, on your plot. d) Now do the same for the enzyme in the presence and absence of I using a Lineweaver-Burke plot. Again, show Km and Vmax for enzyme in the presence and absence of I. We only know what you are thinking from the labels. Plot for c Plot for d e) A Michaelis-Menton enzyme has a Km of 40 µm. What percentage (between 0 and 100) of maximal activity that will be observed at a substrate concentration of 10 µm? Write answer in the provided box to the right f) An enzyme has a kcat of 1000/sec. When a single molecule of enzyme is operating at its maximal rate, how many reactions are occurring per minute?

4 Fa15 BIBC102 final, page 4 5) (14 pts) A HEX upon ye! In class we talked about two isoforms of the enzyme hexokinase. (There are actually 4, but we only spoke about two). One of them is simply called hexokinase, and functions in many tissues. The other form of hexokinase is called glucokinase (or hexokinase IV) and is special. Glucokinase is expressed in only a few tissues, including liver cells and pancreatic β cells. Answer the following questions about these versions of hexokinase, using what you know about them, about enzymes, and the information you get in the questions. a) First, write the reaction that these isoforms catalyze. You are given the substrate glucose. Include the structure of the glucose-derived product. Include other substrates and products as needed. b) One of the key differences between hexokinase (H) and glucokinase (G) is that hexokinase follows Michaelis-Menton kinetics while glucokinase shows cooperative kinetics. Also, hexokinase is half saturated at 1mM, while G is half saturated at 10 mm. Both have same Vmax. With all this information, draw a Vo vs. [glucose] plot, using the axes provided. Indicate which enzyme is associated with each curve. Include the ½ saturation position on the curve for each, Indicate Vmax for each c) One of the enzymes has a Km, and the other does not. Which does, which does not, and why do you say this? (one sentence): d) One of these enzymes can be described by a straight line on a Lineweaver-Burke plot, and the other cannot. Which is which, and why is this the case? (one sentence) e)there are people who produce disease variants of glucokinase with lowered activity. Remembering that this version is expressed in the liver, how would their blood sugar levels compare to normal people? Why? f) There are also people with mutant glucokinase 10 times more active than normal! They have a syndrome called persistent hyperinsulinemic hypoglycemia of infancy (PHHI). They have dangerously low blood sugar, and continuously release too much insulin from their β-cells. Recalling that this hyperactive glucokinase is in the β-cell, why does the over-secretion of insulin in these patients make sense? (One sentence) g) Glucokinase is a monomer, with no quaternary structure. Why is this structural feature strange and not typical? (One sentence)

5 Fa15 BIBC102 final, page 5 6) (11 pts) 12 metabolites walk into a bar The molecules don t change But the questions do. Answer the collection of one-liners below using the letter or letters that are correct. If nothing applies, write NO. A B C D E F G H I J K L 1) A product of liver glucose-6-phosphatase: 2) These are converted into each other by triose phosphate isomerase: 3) First metabolite to donate a phosphate to ADP to make ATP in glycolysis: 4) Produced by enolase in glycolysis 5) Split into two parts by aldolase 6) The first substrate of the pentose phosphate pathway 7) Substrate of pyruvate dehydrogenase complex 8) One of the metabolites involved in the glycerol-phosphate shuttle 9) This molecule is a lactone 10) A product of the glycogen phosphorylase rxn 11) Directly made from one of the products of the glycogen phosphorylase rxn 12) Produced along with NAD + in anaerobic conditions 13) Made from oxaloacetate during gluconeogenesis 14) Made into oxaloacetate during gluconeogenesis 15) Product of glyceraldehyde-3p-dehydrogenase during glycolysis 16) Product of glyceraldehyde-3p-dehydrogenase during gluconeogenesis 17) Released from muscle during intense exercise 18) Substrate of citrate lyase 19) Structure of Fr 2,6 bp 20) Produced by Fr-1,6 bisphosphatase during gluconeogenesis 21) Produced by Fr-1,6 bisphosphatase during glycolysis 7) (1pt) Both gluconeogenesis and glycolysis are exergonic pathways. How can this be true? (it is true; one sentence)

6 Fa15 BIBC102 final, page 6 8) (10 pts) Kanye and Kim and have a Krebaby! To the right is a gallery of molecular structures, all metabolites of the Krebs cycle. Use the letters associated with each structure to answer the following questions. You can use more than one, just one, or NO if no letter is applicable. Give the best answer you can for each. Just like The Four Agreements. 1) Directly produced by the addition of an acetyl group during the Krebs cycle 2) Substrate of an enzyme that catalyzes oxidative decarboxylation 3) Substrate of an enzyme that is very similar to the PDH complex 4) Product of an enzyme that is very similar to the PDH complex 5) The main product of the glyoxyate cycle 6) Not found in the glyoxylate cycle 7) A product of the complex II reaction 8) Substrate that reacts with acetyl-coa to start the Krebs cycle 9) Product of pyruvate carboxylase 10) Its cognate amino acid is alanine 11) Its cognate amino acid is glutamate 12) Its cognate amino acid is aspartate 13) Product of an enzyme that also makes GTP during the reaction 14) Reacts with acetoacetate during utilization of ketone bodies 15) Undergoes enzymatic oxidation by NAD+ 16) Source of acetyl-coa for lipid anabolism 17) One of the products of citrate lyase 18) Produced along with CO 2 during the glyoxylate cycle 19) Involved in the malate-aspartate shuttle as substrates or products 20) Can cross the inner mitochondrial membrane by a transporter 21) Added to fatty acids to facilitate transport to the matrix 22) Substrate of a mutant version of IDH found in 80% of gliomas 9) (2pts) When whole body demand for glucose is high (starvation, endurance events), the liver shows a relative decrease in Krebs cycle molecules and a corresponding over abundance of acetyl- CoA. a) Why are the Krebs cycle molecules depleted? b) Why does this situation favor production of ketone bodies?

7 Fa15 BIBC102 final, page 7 10) (9 pts) Liver the Re-gifter (because I used this last year): The liver is in a very real sense the Minister of Glucose. We have even called the liver a glucostat for the body, since it is so important in keeping blood glucose in a safe range. a) (2 pts) When we have fasted for awhile, and insulin is low and glucagon is high, what happens to the following LIVER processes (a word, phrase, or clause is fine) glycolysis: glycogen synthesis: gluconeogenesis: glycogen breakdown: b) (2 pts) When we have had a meal, and insulin is abundant while glucagon is low, what happens to the following LIVER processes (a word, phrase, or clause is fine) glycolysis: glycogen synthesis: gluconeogenesis: glycogen breakdown: c) (2 pts) The picture shows a key regulatory molecule we learned about in class, called fructose 2,6 bisphosphate. Fr2,6bP is an important part of glucose regulation in the liver. What are its direct effects (phrase fine; none is fine if true) on the following enzymes phospfructokinase 1 (PFK1): fructose 1,6 bisphosphatase : glycogen synthase: glycogen phosphorylase: d) (1 pts) In words (or a word, what are the effects of Fr 2,6bP on these two liver metabolic pathways: Glycolysis: Gluconeogenesis: e) (1 pts) Both glucagon and insulin alter cellular levels of Fr2,6bP in the liver. What is the effect of each hormone on cellular levels of this key regulator of glucose? Glucagon: Insulin: f) (1 pt) Why is glucose produced from glycogen in liver released into the bloodstream, while that produced in muscle is not. Include in your explanation the name of the enzyme that underlies this difference (one sentence)

8 Fa15 BIBC102 final, page 8 11) (17 pts) FAStidiousness Fatty acid synthesis is a critical process in both normal health and the obesity epidemic. The following questions pertain to fatty acid synthesis. a) (1 pt) What is the name of the rate-limiting enzyme for fatty acid synthesis? b) (3 pts) Whether or not you know the name of this key enzyme of fatty acid production, write the reaction that produces malonyl-coa from acetyl-coa, including the substrates and the products, and the cofactors that are needed. I have included acetyl-coa for your test taking convenience. (Structural Hint: malonyl-coa is Often Made from Supplied Glucose) c) (3 pts) Once malonyl-coa is made, it is used to synthesize fatty acids by a multi-activity enzyme complex called fatty acid synthase (FAS). The reaction starts by production of a FAS-linked β-keto acid when malonyl CoA is condensed with acetyl-coa. Using the starting structure shown below, write the reactions that occur to convert this structure into a 4 carbon fatty acyl chain. Include other substrates and products. No enzyme names needed. d) (1 pt) One hears a lot about omega-3 fatty acids. They apparently are good for health in a variety of ways. Draw a generic omega-3 fatty acid (you can use an R for most of the hydrocarbon part). Pay attention to the orientation of the key bond A very interesting connection exists between lipid pathways. It turns out that malonyl-coa is a potent inhibitor of carnitine palmitoyl transferase (CPT) on the outer mitochondrial membrane. e) (1 pt) What is carnitine? : f) (1 pt) What is the enzymatic function of CPT? (words): g) (5 pts) Draw a simple schematic cartoon to the right to demonstrate how the CPTs work in lipid metabolism. Indicate the relevant cellular compartments (eg. cytosol, matrix).you can use R-COOH for fatty acids if needed. h) (2 pt) So, now that you have refreshed your memory, why might the potent inhibition of CPT by malonyl-coa make physiological sense?

9 Fa15 BIBC102 final, page 9 12) (16 pts) The Emperor of All Lipids- We learned in class about the metabolic features of cancer cells. They have two predominant features that we discussed. The metabolic consequences of these special feature may be of use to use in the clinical management of this malady. a) (2 pt) We have discussed the Warburg effect, an observation made about cancer cells before even I was a good idea (eek). What is the Warburg effect (one sentence)? b) (2 pt) In addition to the Warburg effect, cancer cells also have a special metabolic restriction that pertains to fatty acid metabolism. We covered this in class, and there is hope that this metabolic feature may lead to therapeutic approaches. Describe this feature (one sentence) c) (4 pts) Because of the feature in b), there is renewed interest in the enzyme citrate lyase (also called ATP-citrate lyase) as a possible target for cancer. Write the citrate lyase reaction below, using the structure of citrate. Include products derived from citrate as structures, and write the names of other substrates and products. Here s citrate. d) (1 pt) Where does the citrate lyase reaction occur? e) (2 pt) Which two metabolic pathways would be most strongly affected by inhibition of citrate lyase. Just the names. No need to write the pathways 1) 2) f) (3 pts) Normal cells can use fatty acids is by taking them up from the blood and combining them with CoA-SH to allow use in metabolism. This reaction is catalyzed by acyl-coa synthetase. We have discussed this enzyme numerous times. Using the fatty acid provided, complete the acyl-coa synthetase reaction. (Hint: remember what I told you about synthetases ) Just last year, it was reported that cancer cells can use the reaction in f) to attach acetate taken up from the bloodstream to CoA-SH (Comerford et al. Cell Dec 18;159: ). So instead of a big fatty acid group, the substrate is CH 3 -COOH, or acetate. g) (1 pt) What is made by acyl-coa synthetase when acetate is used as the substrate? h) (1 pt) So, why might this reaction make cells less dependent on citrate lyase? (one sentence)

10 Fa15 BIBC102 final, page 10 13) (13 pts) Home-Made Glucose for the Holidays Gluconeogenesis is process by which glucose is made from smaller molecules. We learned about this in great detail in class, and we will discuss it here. a) (2 pts) What tissues can perform gluconeogenesis, and which one is most important in terms of whole body glucose regulation? b) (1.5 pts) Phosphoenoylpyruvate carboxykinase (PEP-CK) is a key enzyme in gluconeogenesis. Write the reaction including the structure of resulting glycolytic metabolite and the names of any other substrates or products. I have included the starting molecule to help, but you need to name it. Remember, this enzyme uses GTP as an energy source (that is called a give ) c) (5 pts) In mammals, gluconeogenesis is an important part of the Cori cycle. In the space provided, draw a cartoon representing the Cori cycle. You can use simple arrow to indicate any pathways; no details required. Show the flow of key compounds to and from, and in and out of, the involved tissues. Keep it simple d) (2 pts) What is the function of the Cori cycle? Why is it important during intense exercise? (one or two sentences) e) (1.5 pts) In mammals, there are three main sources of carbon for gluconeogenesis. (One of them is the metabolite used in the Cori cycle) What are they? 1) 2 3 f) Mammals cannot convert the carbon from fatty acids into glucose, but plants can, and do. 1) (1/2 pt) Why can t mammals use fatty acid carbon to make glucose by gluconeogenesis? (one sentence; please do not say because they don t have the xxxx pathway that is in plants ) 2) (1/2 pt) Why can plants use fatty acid carbon to make glucose by gluconeogenesis? (You may now want to mention the xxxx pathway perhaps) (one sentence)

11 Fa15 BIBC102 final, page 11 14) (8 pts) Acceptance is the key Recently, I was having coffee with world-famous mitochondriac Immo Scheffler. He told me about a paper he published when I was in my senior year of college (right before the Civil War). It is a study of a mutant cell line he discovered: A respiration-deficient Chinese hamster cell line with a defect in NADH-coenzyme Q reductase. J Biol Chem. (1976) 251: Basically it is a description of a mammalian cell line (CHO cells, very common) with a complete deficiency in one of the ETC enzymes, as stated in the title. We will use the features of this paper to explore mitochondrial function. a) (3 pts) First, which ETC complex is this paper about? The enzyme name (in boldface) will tell you. Draw a cartoon in the space of that single ETC complex. Show what it does in your cartoon. Indicate the complex number, the location of the matrix, the inner membrane, show the reaction, and the flow of electrons, and the movement of protons if that happens. Just this one complex please The paper discusses a cell line in which this ETC enzyme complex is totally non-functional due to a mutation. Answer the following questions. b) (2 pts) Write the net reaction for the NADH-coenzyme Q reductase (the complex you just drew above). No structures, just a balanced reaction with the names of substrates consumed and products generated. (I don t care what was on an old test. Make sure your reaction here is balanced.) c) (1 pt) In the mutant cell line, there is a complete lack of NADH-coenzyme Q reductase. What would you expect to happen to NADH levels in the matrix in this circumstance? One sentence. d) (1 pt) The surprising observation in this paper is that the Krebs cycle is totally non functional! There is almost no CO 2 produced from added glucose. In fact, they originally thought the mutation was in a Krebs cycle enzyme, but it is not. The only defective enzyme is the ETC complex member. In fact, the Krebs enzymes are all normally expressed and function fine when isolated from the cell. But when the mitochondrion is intact, the Krebs cycle is essentially stopped. What is going on? Propose a simple explanation for why this ETC mutant causes a shut-down of the Krebs cycle when the cells are intact. It is based on the normal regulation of the Krebs cycle that we have discussed in class. One or sentences e) (1 pt) This mutant shows some of the features of normal regulation of oxidative metabolism. In normal cells, when all the ADP is converted to ATP, a phenomenon called acceptor control occurs. Basically, when ADP is unavailable as a substrate for ATP synthase, both the ETC and the Krebs cycle stop. Why is this? Explain in one or two sentences.

12 Fa15 BIBC102 final, page 12 and to all a good nightrogen! This page is going to include a number of nitrogen-related metabolic ideas. Lets get to it! 14) (10 pts) Urea-ly got me now The urea cycle produces urea, shown in the picture. Answer these questions about the urea cycle a) (2 pts) Why is urea such a smart way to store waste nitrogen? List two characteristics of urea that make it a great way to dispose of nitrogen. 1) 2) b) (2 pts) How is the enzyme carbamoyl phosphate synthetase 1 (CPS-1) important in the urea cycle? One sentence c) (2 pts) since you have the structure of urea, draw the structure of carbamoyl phosphate in the space to the right d) (2 pts) Write the reaction for CPS-1; remember the S stands for synthetase You can just write CP for carbamoyl phosphate since you had to draw it above (no double jeopardy) e) (1 pt) What is N-acetyl-glutamate (NAG)? (Hint: it is conceptually similar to Fr 2,6 bp) f) (1 pt) People born with a defect N-acetyl-glutamate synthase (NAGS) suffer from severe nitrogen toxicity with a number of early symptoms observed in the neonate. Why does this make sense? 15) (3 pts) Home bases This is a brief question about nucleotides and such. Almost done! a) What are the two main chemical differences between RNA and DNA. One phrase or sentence each. I am NOT looking for RNA is used to translate proteins, and DNA stores the genetic information. Rather, I am looking for things we talked about in this class. eg RNA uses glucose and DNA uses fructose in its structure ; that would be wrong, but it is the right idea) 1): 2): b) Lesch-Nayhan disease tells us that a particular aspect of nucleotide metabolism is very important. What is the process, and why does the disease tell us that (one sentence)

13 Fa15 BIBC102 final, page 13 16) (14 pts) True False Answer T or F for each; best answer a) ketone bodies are produced in the liver b) ketone bodies are consumed by the liver c) ketone bodies are produced in muscle d) ketone bodes are consumed by muscle e) glucose can be made from some breakdown products of triglycerides f) arachidonic acid is an essential fatty acid g) arachidonic acid is a substrate of cyclooxygenase h) gout is caused by excess production of uric acid i) the enzyme blocked in the treatment of gout uses PRPP as a substrate j) muscles produce alanine from pyruvate to facilitate removal of ammonia k) insulin signaling is hypothesized to promote aging l) there are genes that normally function to diminish lifespan m) there are genes that normally function to increase lifespan n) gluconeogenesis is only regulated by glucagon o) Fr2,6bP levels are controlled by a single enzyme with two catalytic activites p) Keq varies with concentrations of reactants or products q) ΔG o determines how fast a reaction will occur r) ΔG determines how fast a reaction will occur s) The pentose phosphate pathway is important for fatty acid synthesis t) Type I muscle fibers are far more abundant in elite endurance runners u) anaerobic metabolism delivers more power to muscles v) Type I fibers obtain most of their ATP through glycolysis alone w) After extended exercise, glucagon is high and insulin is low x) You have come to the end of your metabolite road.