Name: Chem 465 Biochemistry II Test 1 Spring 2018 Multiple choice (4 points apiece): 1. During strenuous exercise, the NADH formed in the glyceraldehyde 3-phosphate dehydrogenase reaction in skeletal muscle must be reoxidized to NAD+ if glycolysis is to continue. The most important reaction involved in the reoxidation of NADH is: A) dihydroxyacetone phosphate 6glycerol 3-phosphate B) glucose 6-phosphate 6 fructose 6-phosphate C) isocitrate 6 á-ketoglutarate D) oxaloacetate 6malate E) pyruvate 6 lactate 2. If glucose labeled with 14C in C-3 is metabolized to lactate via fermentation, the lactate will contain 14C in: A) all three carbon atoms. B) only the carbon atom carrying the OH. C) only the carboxyl carbon atom. D) only the methyl carbon atom. E) the methyl and carboxyl carbon atoms. 3. Which of the following is not true of the citric acid cycle? A) All enzymes of the cycle are located in the cytoplasm, except succinate dehydrogenase, which is bound to the inner mitochondrial membrane. B) In the presence of malonate, one would expect succinate to accumulate. C) Oxaloacetate is used as a substrate but is not consumed in the cycle. D)Succinate dehydrogenase channels electrons directly into the electron transfer chain. E) The condensing enzyme is subject to allosteric regulation by ATP and NADH. 4. The reaction of the citric acid cycle that produces an ATP equivalent (in the form of GTP) by substrate level phosphorylation is the conversion of: A) citrate to isocitrate. B) fumarate to malate. C) malate to oxaloacetate. D) succinate to fumarate. E) succinyl-CoA to succinate. 5. Which of the following cofactors is required for the conversion of succinate to fumarate in the citric acid cycle? A) ATP B) Biotin C) FAD D) NAD+ E) NADP+ 6. (20 points) This page is blank because I want you to fill it in with the glycolytic pathway from glucose to pyruvate showing the structure of all intermediates. At each step also give the name of the enzyme and ÄG of the reaction. To help you on your way, here is a list of all then enzymes in alphabetical order: Aldolase Enolase Glyceraldehyde 3-phosphate dehydrogenase Hexokinase Phosphofructokinase-1 Phosphoglycerate kinase Phosphoglycerate mutase Phosphohexose isomerase Pyruvate kinase Triose phosphate isomerase Also show the reactions and enzymes involved in gluconeogenesis On this key I am not going to take the time to make the structures, but I will check that you have the correct structure Glucose + ATP Hexokinase Mg2+ -16.7 Glucose-6-P Phosphohexose isomerase Mg2+ +1.7 Fructose-6-P + ATP Phosphofructosekinase -1 Mg2+ -14.2 Fructose 1,6-bisphosphate aldolase +23.8 Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate Triose isomerase +7.5 Glyceraldehyde 3phosphate + NAD glyceraldehyde 3-phosphate dehydrogenase + 6.3 1,3-Bisphosphoglyerate phosphoglycerate kinase Mg2+ -18.5 3-Phosphoglycerate + ATP Phosphoglyerate mutase Mg2+ +4.4 2-Phosphoglycerate Enolase Mg2+ +7.5 Phophoenolpyruvate Pyruvate kinase K+, Mg2+ or Mn2+ -31.4 Pyruvate + ATP -2- 7. (20 points) Sketch the pathway in which pyruvate is completely oxidized to CO2. In this pathway be sure to identify all reactions that generate ATP, GTP, NADH or FADH2. Also show the anapleotropic reaction that are used to fill in TCA intermediates when they are depleted. I would prefer structures for all intermediates, and a cycle, but this is something I can’t put in with a word processor Pyruvate pyruvate dehydrogenase CoASH,NAD, TPP,Lippoate, FAD -33.3 Acetyl CoA (Oxaloacetate) citrate syntase -32.2 Citrate aconitase Iron-sulfur center 13.3 Isocitrate + NAD 6NADH isocitrate dehydrogenase Mn2+ NAD -20.9 á-ketoglutarate + NAD 6NADH á-ketoglutarate dehydrogenase CoASH,NAD, TPP,Lippoate, FAD -33.5 Succinyl CoA + GDP or ADP 6GTP or ATP Succinyl-CoA synthetase -2.9 6 Succinate + FAD FADH2 Succinate dehydrogenase FAD, FeS clusters 0 Fumarate fumarase -3.8 L-malate + NAD 6NADH l-malate dehydrogenase 29.7 Oxaloacetate Anapleotropic: - 6 Pyruvate + HCO3 + ATP Oxaloacetate + ADP + Pi pyruvate carboxylase 6 PEP + CO2 + GDP oxaloacetate + GTP PEP carboxykinase - 6 PEP + HCO3 oxaloacetate + Pi PEP carboxylase - 6 Pyruvate + HCO3 + NADH malate + NAD malic enzyme -3- On the following pages are 6 questions, each question is worth 10 points. Chose and 4 questions. 8. Describe the regulatory role of fructose 2,6-biphosphate. What signals change the concentrations of fructose 2-6 biphosphate, and how do these changes effect the glycolytic/gluconeogenic pathway. Fructose 2,6 Bisphosphate increases the activity of Phosphofructosekinase which increases the overall rate of glycolysis and inhibits gluconeogenesis. In the absence of F26BP the glycolytic pathway is slowed and gluconeogenesis is stimulated. The levels of F26BP are in turn controlled by the hormones glucagon and insulin. Glucagon increases the amount of cAMP in the cell. This increases the activity of cAMP dependent protein kinase which phosphorylates the PFK-2/FBPase-2 enzyme, deactivation the PFK-2 activity amd activation the FBPase-2 activity. The FBPase-2 dephosphorylates Fructose 2,6 bisphosphate lowering the concentration of this signal molecule inhibiting glycolysis and stimulating gluconeogenesis. Insulin increases the activity of phosphoprotein phosphatase with removes the phosphate from the PFK-2/FBPase-2 enzyme. This turns on the PFK-2 activity and turns off the FBPase-2 activity. The levels of F26BP rise, glycolysis is stimulated and gluconeogenesis is inhibited. Overall Insulin is the signal that the body has enough glucose so it stimulates the destruction of glucose while glucagon is the signal that glucoe levels are too low, and the body tries to make more glucose 9. Pyruvate dehydrogenase complex, á-ketoglutarate complex and the complex that oxidizes branched hydrophobic amino acids all have a very similar overall structure. Outline the overall structure of this complex and how it is similar or different between three different complexes. A diagram like figure 16-6 is a great start. These complexes are massive, more than 5 times the size of the ribosome. Typically they involve 3 major components, E1, E2 and E3. In the bovine system there are 60 copies of E2 that make a E3 are wedges between the E2's while E1 is around the outside. The E1's bind the pyruvate, á-ketoglutarate or amino acid and the coenzyme TPP. Because they bind the starting substrate they have very similar structure but vary in the substrate binding site. The E2's contain the lipoic acid covalently attached to a lysine and bind the Coenzyme A. The structures of the E2, vary a little bit from complex to compex. The E3's contain the FADH and bind the NADH, and are virtually idential between the different complexes -4- 10. Outline the major controls for the TCA cycle. Although not technically part of the TCA cycle, pyruvate dehydrogenase complex which makes the acetyl-CoA that feed into the cycle is inhibited by ATP, acetyl-CaA, NADH, and fatty acids and stimulated by AMP, free CoA, NAD+ and Ca2+. Citrate Synthase, the first enzyme in the cycle is inhibited by NADH, succinyl- CaA, citrate and ATP. It is stimulated by ADP. Isocitrate dehydrogenase is stimulated by Ca2+ and ADP, but inhibited by ATP. á-ketoglutarate dehydrgenase is inhibited by succinyl-CoA and NADH and stimulated by Ca2+ 11. What is ROS, why is it bad, and how do you get rid of it. ROS stands for Reactive Oxygen Species and includes free radical species like @ - @ O2 , OH. These free radicals are very reactive and tend to react with anything they touch including proteins and DNA, and thus can really damage a cell if they aren’t eliminated quickly. A figure like 19-18 helps here. @ - O2 is first reacted with protons to make H2O2 by the enzyme superoxide dismutase. The hydrogen peroxide is then used to oxidize 2 glutatione molecules (GSH) into a single GSSG molecule by glutathione peroxidase. Glutathione reductase then uses NADPH to reduce the GSSG back to 2 GSH molecules. 12. Glutamic acid can enter the TCA cycle as á-ketoglutarate. If glutamic acid is labeled on the á-carbon (see structure below) where does that carbon end up in isocitrate after 1 turn of the TCA cycle. (Hint: look at your answer to problem 7 to help you trace where the label goes.) The label in this glutamic acid is in the á position, between the NH2 and the COOH. Glutamic acid undergoes a transamination reaction to become á- ketoglutarate labeled in the ketone. In the next step the COOH next to the label is removed and the labeled Carbon is attached to the S-Co-A in succinyl-CoA. In the next step when the S-CoA is removed the molecule becomes the symmetric Succinic acid (Succinate) and the label is on one of the two COOH positions. Following the cycle around the label and end up at either of the COOH’s on Oxaloacetate. From here it ends up at either the middle or bottom COOH in citric acid and finally in the middle or bottom COOH in Isocirate -5- 13. ATP is made in the mitochondria, yet it must make its was to the cytosol where it is used and converted into ADP and Pi. Describe the transport systems that get ATP, ADP and Pi into, and out, of the mitochondira. Are these passive or active transport systems, and, if active transport, what is the energy source of the active transport. Here you need a figure like figure 19-30. You need two different transporters. One is a passive antiport system that brings 1 ADP into the mitochondria as it lets 1 ATP out. Assuming the ATP concentration in higher inside the mitochondira than outside, and ADP is higher outside than inside no additional energy is neede for transport.