Name: 2 points Chem 465 Biochemistry II Test 1 Spring 2016 Multiple choice (4 points apiece): 1. The steps of glycolysis between glyceraldehyde 3-phosphate and 3-phosphoglycerate involve all of the following except: A) ATP synthesis. B) catalysis by phosphoglycerate kinase. C) oxidation of NADH to NAD+. D) the formation of 1,3-bisphosphoglycerate. E) utilization of Pi. 2. Which one of the following statements about gluconeogenesis is false? A) For starting materials, it can use carbon skeletons derived from certain amino acids. B) It consists entirely of the reactions of glycolysis, operating in the reverse direction. C) It employs the enzyme glucose 6-phosphatase. D) It is one of the ways that mammals maintain normal blood glucose levels between meals. E) It requires metabolic energy (ATP or GTP). 3. Aside from maintaining the integrity of its hereditary material, the most important general metabolic concern of a cell is: A) keeping its glucose levels high. B) maintaining a constant supply and concentration of ATP. C) preserving its ability to carry out oxidative phosphorylation. D) protecting its enzymes from rapid degradation. E) running all its major metabolic pathways at maximum efficiency. 4. Glucose labeled with 14C in C-3 and C-4 is completely converted to acetyl-CoA via glycolysis and the pyruvate dehydrogenase complex. What percentage of the acetyl-CoA molecules formed will be labeled with 14C, and in which position of the acetyl moiety will the 14C label be found? A) 100% of the acetyl-CoA will be labeled at C-1 (carboxyl). B) 100% of the acetyl-CoA will be labeled at C-2. C) 50% of the acetyl-CoA will be labeled, all at C-2 (methyl). D) No label will be found in the acetyl-CoA molecules. E) Not enough information is given to answer this question. 5. In mammals, each of the following occurs during the citric acid cycle except: A) formation of á-ketoglutarate. B) generation of NADH and FADH2. C) metabolism of acetate to carbon dioxide and water. D) net synthesis of oxaloacetate from acetyl-CoA. E) oxidation of acetyl-CoA. 6. Which of the following is correct concerning the mitochondrial ATP synthase? A) It can synthesize ATP after it is extracted from broken mitochondria. B) It catalyzes the formation of ATP even though the reaction has a large positive G'°. C) It consists of F0 and F1 subunits, which are transmembrane (integral) polypeptides. D) It is actually an ATPase and only catalyzes the hydrolysis of ATP. E) When it catalyzes the ATP synthesis reaction, the G'° is actually close to zero. 7. The relative concentrations of ATP and ADP control the cellular rates of: A) glycolysis. B) oxidative phosphorylation. C) pyruvate oxidation. D) the citric acid cycle. E) all of the above. Longer questions (14 points each) - You may skip any 1 question. 8. Discuss the different levels of control that help to balance phosphofructokinase-1 in the glycolytic pathway and fructose 1,6- bisphoshatase-1 in the gluconeogenesis pathway. ATP is an allosteric inhibitor of Phosphofructokinase (PFK-1), and act as an inhibitor by lowering affinity for substrate. ADP and AMP act to relieve this inhibition. Citrate (from TCA cycle) acts to enhance inhibitory effect of ATP 1,6 bisphosphatase is allosterically inhibited by AMP Additional hormonal control through fructose 2,6-Bisphophate when blood glucose too low, glucagon released this releases cAMP in cell This stimulates cAMP dependent protein kinase This phosphorylates the PFK-2/FBPase-2 enzyme changing it to the form where the FBPase-2 is active and PFK-2 is inactive This lowers level of fructose 2,6-Bisphosphate This allosterically makes PFK-1 almost completely inactive, so glycolysis slows to a standstill, and it activates 1,6- bisphoshatase-1 to turn on gluconeogenesis. Conversely when blood sugar is high, insulin is released this increases the activity of phosphoprotein phosphatase which de- phosphorylates the PFK-2/FBPase-2 enzyme changing it to the form where the FBPase-2 is inactive and PFK-2 is active This raises the concentration of fructose 2,6-Bisphophate This stimulates PFK-1 and inhibits FBPase-1 increasing glycolysis and shutting down gluconeogenesis. -2- 9. This page is blank because I want you to fill it in with the gluconeogenesis pathway from pyruvate to glucose by 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, Fructose 1,6-bisphosphatase-1, Glucose 6-phosphatase, Glyeraldehyde 3-phosphate dehydrogenase, PEP carboxykinase, Phosphoglycerate kinase, Phosphoglycerate mutase, Phosphohexose isomerase, Pyruvate carboxylase, Triose phosphate isomerase. I know you were expecting the glycolytic pathway. If you need to, do the glycolytic pathway and then put in the reverse arrows and the appropriate reverse reactions and change the sign of the ÄG’s. Don’t worry about the ÄG’s of the 4 reverse reactions that I didn’t tell you to memorize. - 6 Pyruvate + HCO3 + ATP oxaloacetate + ADP + Pi ÄG ? Pyruvate carboxykase Biotin oxaloactate + GTP 6PEP + GDP ÄG? Phosphoenolpyruvate carboxykinase Mg2+ Two PEP 6 two 2-Phosphoglycerate ÄG -7.5 Enolase Mg2+ Two 2-Phosphoglycerate6Two 3-Phosphoglycerate ÄG -4.4 Phosphoglycerate mutase Mg2+ Two 3-Phosphoglycerate + 2 ATP6Two 1,3-Bisphophoglycerate + 2ADP ÄG +18.5 phosphoglycerate kinase Mg2+ Two 1,3-Bisphosphoglycerate +2 NADH6Two Glyceraldehyde-3-P + 2NAD+ ÄG -6.3 glyceraldehyde 3-phosphate dehydrogenase One Glyceraldehyde-3-P 6One Dihydroxyacetonephosphate ÄG -7.5 Triose isomerase Dihydroxyactone phosphate + Glyceraldehyde-3-P 6Fructose 1,6bisphosphate Aldolase ÄG -23.8 Fructose-1,6-bisphophate6Fructose-6-P + Pi ÄG -16.3 Fructose 1,6-bisphophatase Mg2+ Fructose 6-P6Glucose 6-P ÄG -1.7 Phophohexose isomerase Mg2+ Glucose 6-P6Glucose ÄG -13.8 Glucose 6-phophatase Mg2+ -3- 10. 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 -4- 11. Discuss how the pyruvate dehydrogenase complex works, and how it is similar to or different than the á-ketoglutarate dehydrogenase complex. Include names of all cofactors involved in the complexes and the steps involved in the overall reaction. I would start with a sketch like figure 16-6 from your text. The PDH complex contains three enzymes, Pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3) The number of copies of each enzyme varies from species to species, but hey are all associated into one big complex that can be 5x bigger than a ribosome. The pyruvate is first decarboxylated on E1 and the remaining 2 carbons are bound to Thiamine pyrophosphate on this enzyme as a hydroxyethylgroup. This hydroxyethylgroup is then passed as an acetyl group to a lipoyllysine on the E2 enzyme. The acetyl group is then passed to Coenzyme A, leaving the lipoyllysine in a reduced form. The reduced lipoyllysine is then reoxidized by an FAD group on the E3 enzyme + which get reduced to FADH2, and then the FADH2 is reoxidized by an NAD that gets reduced to NADH on the E3 enzyme The pyruvate dehydrogenase and the á-ketoglutarate enzymes are very similar in both the E2 and the E3 parts of the complex. The main difference lies in the E1 part of the complex since it must interact with a different substrate. -5- 12A. If you start with 1 NADH in the mitochondrial matrix How many protons are transported out of the mitochondrial matrix in Complex I? __4_ How many protons are transported out of the mitochondrial matrix in Complex II? __0__ How many protons are transported out of the mitochondrial matrix in Complex III? _4___ How many protons are transported out of the mitochondrial matrix in Complex IV? _2___ 12 B. Depending on the organism, ATP synthase can have between 8 to 15 c subunits in the FO complex, and this, in turn, determines the number of protons that come back into the mitochondrial matrix for one complete turn of synthase complex. Based on your answer for 12A, how many ATPs are synthesized from one NADH in an organism with 9 c subunits in the Fo complex (you may ignore the ATP used in transport of Pi)? Explain. With 9 c units it takes 9 protons to make the ATPsynthase do a complete turn. Since 1 turn generates 3 ATP there are 9 protons/3 ATP Our NADH generated 10 protons so 10 protons x 3 ATP/9 protons = 3.3 ATP /NADH Based on your answer for 12A, how many ATPs are synthesized from one NADH in an organism with 15 c subunits in the Fo complex (you may ignore the ATP used in transport of Pi)? Explain.