Name: Chem 465 Biochemistry II Test 1 Spring 2019 Multiple choice (4 points apiece): 1. In an anaerobic muscle preparation, lactate formed from glucose labeled in C-3 and C-4 would be labeled 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. 2. In an anaerobic muscle preparation, lactate formed from glucose labeled in C-2 would be labeled 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. All of the following enzymes involved in the flow of carbon from glucose to lactate (glycolysis) are also involved in the reversal of this flow (gluconeogenesis) except: A) 3-phosphoglycerate kinase. B) aldolase. C) enolase. D) phosphofructokinase-1. E) phosphoglucoisomerase. 4. Cellular isozymes of pyruvate kinase are allosterically inhibited by: A) high concentrations of AMP. B) high concentrations of ATP. C) high concentrations of citrate. D) low concentrations of acetyl-CoA. E) low concentrations of ATP. 5. Which of the following is not true of the reaction catalyzed by the pyruvate dehydrogenase complex? A) Biotin participates in the decarboxylation. B) Both NAD+ and a flavin nucleotide act as electron carriers. C) The reaction occurs in the mitochondrial matrix. D) The substrate is held by the lipoyl-lysine "swinging arm." E) Two different cofactors containing -SH groups participate. 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 Reverse These reactions not as well described so no ÄG’s or cofactors - Pyruvate +ATP+ HCO3 Pyruate carboxylase Oxaloacetate + GTP PEP carboxylase PEP 6 Fructoae 1,6-bisphosphate fructose 1,6-bisphosphatase-1 Glucose 6-phosphate glucose 6-phosphatase -2- 10 point questions - You may skip one! 7. What is the difference between: ‘Triphosphate and a ‘trisphosphate’ Triphosphate - 3 phosphates attached in a line at one spot Trisphosphate -3 phosphates attached at different places Metabolic control and metabolic regulation Metabolic control -mechanism that allow an organism to change with changing situation Metabolic regulation - mechanisms that try to maintain homeostasis A kinase and a phosphatase Kinases add phosphates, phosphatases remove phosphates A substrate cycle and a futile cycle A cycle of one enzyme doing the reaction of A6B, and a second enzyme doing the reaction B6A. Substrate cycling is used to refer to a low level of this cycling that seems to go on in all cells, Futile cycling refers to doing this at a high level that would waste energy and be damaging t the cell. PFK-1 and PFK-2 Phosphofructokinase 1 and 2. 1 is used in glycolysis and makes Fructose 1,6- bisphosphate, 2 us used in control and makes Fructose 1,6 bisphosphate. 8. I told you that you didn’t have to worry about most mechanisms for most of the enzymes except one, the mechanism of phosphoglycerate mutase. What reaction does this enzyme catalyze, why was this mechanism ‘special’, and what other enzyme shares a similar mechanism? The reaction is 3-phosphoglycerate to 2-phosphoglycerate. The unique feature of the mechanism is that it does not simply mover the same phosphate from one carbon to the other. What is actually does is to take a phosphate already on a histidine of the enzyme and add it to the 3-phosphoglycerate to make 2,3-bisphosphoglycerate, and then that same histidine is used to remove the phosphate on the 3 position to make the final product and regenerate the phosphoenzyme. The enzyme with a similar mechanism is succinyl-CoA synthetase in the TCA cycle. This enzyme first puts an inorganic phosphate on a histidine on the enzyme as it releases CoA from succinyl- CoA, then this phosphate if attached to GDP to make GTP. 9. The pentose phosphate pathway is divided into two processes, the oxidation of glucose-6-phosphate to Ribose-5-phosphate, and the non-oxidative conversion of ribose-5-phosphate to Glucose-6 phosphate. Describe in general terms what is going in these two different processes and why each process is important. In the first oxidative part of the pathway glucose 6-phosphate is oxidatively decarboxylated to Ribose 5-phosphate a 5 carbon sugar. In this process 2 NADPH’s are generated the can be used in synthetic pathways. If this pathwau produces too many Ribose sugars, in the second part of the pathway six of the 5-carbon sugars are converted into five 6-carbon sugars . -3- 10. In Chapters 14 and 15 I have often shortened chemical and enzyme names to a simple three or four character code. Decode the following: (1 point each - bonus point if you get all 11 right) Example: ATP - Adenosine triphosphate Proteins Chemicals PFK-1 F26BP Phosphofructokinase-1 Fructose 2,6-bisphosphate FBPase-2 PEP Fructose bisphosphatase Phosphoenolpyruvate GLUT4 AMP Glucose Transporter-4 Adenosine monophosphate LDH NADH Lactate dehydrogenase Nicotinamide adenine dinucleotide G6P Glucose 6-phosphate DHAP Dihydroxyacettone phosphate G3P Glyceraldehyde 3-phosphate 11. Under different conditions (aerobic or anaerobic) different organisms do different things with pyruvate and the end of glycolysis. Describe the different things organisms do with pyruvate and why each of these different fate is advantageous to the organism. Aerobic organism under aerobic conditions Pyruvate enter the mitochondria and is completely oxidized to CO2 This pathway delivers the maximum energy to the organism Aerobic organism under anaerobic conditions Pyruvate is reduced to Lactictic acid This pathway is used when the TCA cycle in the miitochondria is shut down due to lack of oxygen. Under these circumstances the NADH produced during the oxidation of glyceraldehyde 3-phosphate must be changed back into NAD+ otherwise the glycolytic pathway shuts down due to lack of NAD+ and the organism has no energy sources. The lactic acid produced here can be changed back to pyruvate for further oxidate or the pyruvate can be changed back into glucose via gluconeogenesis. Anaerobic organisms Pyruvate is converted first to acetaldehyde and then to ethanol. Because the conversion of acetaldehyde to ethanol uses NADH, this is an alternate pathway to regenerate the NAD+ needed in the glycolytic pathway. Unlike lactic acid the final product of ethanol cannot be changed back to glucose via gluconeogenesis. -4- 12. Describe the controls used to regulate the conversion of glucose to glucose 6- phosphate in glycolysis and glucose 6-phosphate to glucose in gluconeogenesis. This set of enzymes, hexokinase on the ‘forward’ direction and glucose-6-phosphatase in the ‘reverse’ direction is under fairly simple control. In the ‘backward’ direction the phosphatase is only found in specific liver, kidney, and intestinal cells, where the glucose created in this reaction can be delivered to other cells by the bloodstream. This also prevents other cells that can use Glucose 6- phosphate more readily than glucose from setting up a futile cycle between the hexokinase and the phosphatase. In the ‘forward’ direction there are different tissue specific controls. Muscle cells have primarily Hexokinase I and II. In these cells the kinase is fully active at glucose concentrations just slightly above .2 mM, since the normal fasting level of glucose in the blood it 5 mM, this forward reaction is completely turned on in muscle. Hexokinase I & II are also allosterically inhibited by the product of this reaction, glucose 6-phosphate, so this reaction is slowed if the glycolytic pathway is not being used so G 6-P builds up. The liver isozyme (Hexokinase IV or glucokinase) behaves differently. This isozyme is only fractionally turned on at the fasting blood sugar level of 5 mM, and is not inhibited by G 6-P so glucose levels can build up in the liver, and this glucose can leave the liver cell, and enter the bloodstream to get transported to other cells. The liver hexokinase has an additional control in that there is a regulatory protein that binds to the hexokinase and moves it to the nucleus. This protein is activated to transport the hexokinase out of the cytosol when the levels of Fructose 6-phosphate rise (indicating the glycolytic pathway is being shut down) and the protein releases the hexokinase back into the cytosol when glucose is preset. Both the kinase and the phosphatase are subject to additional transcriptions control that were not discussed in class. 13. The Pyruvate dehydrogenase complex requires five non-protein coenzymes or cofactors to function. Name these five coenzymes, and describe briefly what chemistry they do.