2011 BIOC221

Problem Set (3)

Due date: Feb 8 (Tue, 11:30 am)

1. Glucose Transporters Read Lehninger p. 391393, p. 539, and p. 542. Glucose enters cells by facilitated diffusion via a specific glucose transporter, at a rate ~5000 times greater than uncatalyzed transmembrane diffusion. The transporter is, in principle, equally able to move glucose into or out of the cell. Since the glucose conc. in blood plasma is ~5 mM and the intracellular glucose conc. is kept low (owing to fast metabolism of glucose), glucose always moves down its conc. gradient (thus, passive transport). Human genome contains 12 glucose transporter genes. Each transporter shows unique specificity toward different hexose, different pattern of tissue distribution, and function (see Table 11-3, p. 391). Compare the localization of GLUT4 with that of GLUT2 and GLUT3, and explain why these localizations are important in the response of muscle, adipose tissue, brain, and liver to insulin. Answer GLUT2 (and GLUT1) is found in liver and is always present in the plasma membrane of liver cells (hepatocytes). GLUT3 is always present in the plasma membrane of certain brain cells. GLUT4 is normally sequestered in vesicles in cells of muscle and adipose tissue and enters the plasma membrane only in response to insulin. Thus, liver and brain can take up glucose from blood regardless of insulin level, but muscle and adipose tissue take up glucose only when insulin levels are elevated in response to high blood glucose. This makes sense because muscle and adipose tissues, being not vital, are the tissues that should get glucose only when it is abundant. 2. Pathway of Atoms in Fermentation (1) A pulse-chase experiment using 14C-labeled carbon sources is carried out on a yeast extract maintained under strictly anaerobic conditions to produce ethanol. The experiment consists of incubating a small amount of 14C-labeled substrate (the pulse) with the yeast extract just long enough for each intermediate in the fermentation pathway to become labeled. The label is then chased through the pathway by the addition of excess unlabeled glucose. The chase effectively prevents any further entry of labeled glucose into the pathway. (a) If [1-14C]glucose (glucose labeled at C-1 with 14C) is used as a substrate, what is the location of 14C in the product ethanol? Explain. (Hint: Handout(3), p. 11 and p. 24, or Fig. 14-6) (b) Where would 14C have to be located in the starting glucose to ensure that all the 14C activity is liberated as 14CO2 during fermentation to ethanol? Explain. Answer Anaerobiosis requires the regeneration of NAD+ from NADH in order to allow glycolysis to continue. (a) Figure 146 illustrates the fate of the carbon atoms of glucose. C-1 (or C-6) becomes C-3 of glyceraldehyde 3-phosphate and subsequently pyruvate. When pyruvate is decarboxylated and reduced to ethanol, C-3 of pyruvate becomes the C-2 of ethanol (14CH3CH2OH). (b) If all the labeled carbon from glucose is converted to 14CO2 during ethanol fermentation, the original label must have been on C-3 and/or C-4 of glucose, because these are converted to the carboxyl group of pyruvate.

3. Pathway of Atoms in Fermentation (2) Through the magic of time travel, you find yourself working as a scientist in Arthur Hardens group. (For who Harden is, see Lehninger p.531.) You found yourself trying to convince Harden that the product of glycolysis, pyruvate, can be converted to lactic acid when no oxygen is available. Hardens group has the expertise to detect lactic acid, and so to prove your point you radiolabel carbon four of glucose and feed this to a microorganism under anaerobic conditions. See p. 24 of Handout (3), or Lehninger p. 547 for help, if needed. (a) When you isolate lactic acid, which carbon do you find has been radiolabelled? (b) To Hardens surprise, only half the lactic acid produced contains radiolabel, but this is not a mystery to you. Explain. (c) Harden is now really excited about this new technique, and therefore conducts the same experiment under aerobic conditions later that evening. To his complete surprise, the radiolabel completely disappears from the system. When you return in the morning, you can explain his results. What is your explanation? Answer (a) The C-4 radiolabel of glucose ends up as C-1 of lactate (the carboxyl group). (b) Half the glucose is split into glyceraldehyde-3-P and half into dihydroxyacetone phosphate, which is isomerized to glyceraldehyde-3-P. C-4 will end up as part of the glyceraldehyde-3-P, while the other glyceraldehyde-3-P will originate from dihydroxyacetone phosphate that would have contained no label. Since glyceraldehyde-3-P, is eventually converted to pyruvate and finally lactate, only half the lactate will be radiolabelled (1mark). (c) The carbon originating from C-4 will be immediately lost as CO2 in the pyruvate decarboxylase reaction, and therefore has bubbled out of the reaction mixture. A gas trap would be required to isolate the radiolabel. 4. Glycolysis There are two reactions in glycolysis in which an aldose is isomerized to a ketose. For
one of these reactions draw the structures of the aldose and the ketose. For both reactions the G' is positive. Briefly explain how the reactions are able to proceed without the input of additional energy. Ans: The two reactions are those catalyzed by phosphohexose isomerase and triose phosphate isomerase: glucose 6-phosphate fructose 6-phosphate (aldose) (ketose) dihydroxyacetone phosphate glyceraldehyde 3-phosphate (ketose) (aldose) Although both of these reactions have standard free-energy changes ( G') that are positive, they can occur within cells because the products are immediately removed by the next step in the pathway. The result is a very low steady-state concentration of the products, making the actual free-energy changes (G) negative: G = G' + RT ln ([products]/[substrates]) In other words, the rxn is driven forward by mass action.

5. Glycolysis Define fermentation and explain, by describing relevant reactions, how it differs from glycolysis. Your explanation should include a discussion of the role of NADH in the reaction(s). Ans: Fermentation is the operation of the glycolytic pathway under anaerobic conditions. Under aerobic conditions, the pyruvate produced by glycolysis is oxidized to acetyl-CoA, which passes through the citric acid cycle. NADH produced in the oxidations passes electrons to O2, and is thus recycled to NAD+ allowing the continuation of the glycolytic reactions. When no O2 is available to reoxidize the NADH produced by the glyceraldehyde 3-phosphate dehydrogenase reaction, electrons from NADH must be passed to one of the products of glycolysis, such as pyruvate or acetaldehyde, forming lactate or ethanol. 6. Glycolysis Shortcut Suppose you discovered a mutant yeast whose glycolytic pathway was shorter because of the presence of a new enzyme catalyzing the reaction Glyceraldehyde 3-P + H2 + NAD+ 3-phosphoglycerate + NADH + H+ Would shortening the glycolytic pathway in this way benefit the cell? Explain. Consider metabolic outcomes only in anaerobic conditions. Answer Under anaerobic conditions, the phosphoglycerate kinase and pyruvate kinase reactions are essential. The shortcut in the mutant yeast would bypass the formation of an acyl phosphate by glyceraldehyde 3-phosphate dehydrogenase and therefore would not allow the formation of 1,3-bisphosphoglycerate. Without the formation of a substrate for 3phosphoglycerate kinase, no ATP would be formed. Under anaerobic conditions, the net reaction for glycolysis normally produces 2 ATP per glucose. In the mutant yeast, net production of ATP would be zero and growth could not occur. Under aerobic conditions, however, because the majority of ATP formation occurs via oxidative phosphorylation, the mutation would have no observable effect. 7. Mechanism Propose a mechanism for the conversion of dihydroxyacetone phosphate into glyceraldehyde 3-P by triose phosphate isomerase based on the mechanism of phosphoglucose isomerase that catalyzes the conversion of glucose 6-P into fructose 6-P. Indicate any general acid/base group at the enzyme active site that is involved in the catalysis. Answer The mechanism is analogous to that for phosphoglucose isomerase. It proceeds through an enediol intermediate. The active site would be expected to have a general base and a general acid. H
B H C OH HA C O CH2OP BH H C OH C OH CH2OP enediol intermediate A H C O HC OH CH2OP