Name: 2 points Chem 465 Biochemistry II Test 1 Spring 2017

Multiple choice (4 points apiece):

1. Which of the following statements about the oxidative decarboxylation of pyruvate in aerobic conditions in animal cells is correct? A) One of the products of the reactions of the pyruvate dehydrogenase complex is a thioester of acetate. B) The methyl (-CH3) group is eliminated as CO2. C) The process occurs in the cytosolic compartment of the cell. D) The pyruvate dehydrogenase complex uses all of the following as cofactors: NAD+, lipoic acid, pyridoxal phosphate (PLP), and FAD. E) The reaction is so important to energy production that pyruvate dehydrogenase operates at full speed under all conditions.

2. In comparison with the resting state, actively contracting human muscle tissue has a: A) higher concentration of ATP. B) higher rate of lactate formation. C) lower consumption of glucose. D) lower rate of consumption of oxygen E) lower ratio of NADH to NAD+.

3.The metabolic function of the pentose phosphate pathway is: A) act as a source of ADP biosynthesis. B) generate NADPH and pentoses for the biosynthesis of fatty acids and nucleic acids.

C) participate in oxidation-reduction reactions during the formation of H2O. D) provide intermediates for the citric acid cycle. E) synthesize phosphorus pentoxide.

4. Gluconeogenesis must use "bypass reactions" to circumvent three reactions in the glycolytic pathway that are highly exergonic and essentially irreversible. Reactions carried out by which three of the enzymes listed must be bypassed in the gluconeogenic pathway? 1) Hexokinase 2) Phosphoglycerate kinase 3) Phosphofructokinase-1 4) Pyruvate kinase 5) Triosephosphate isomerase

A) 1, 2, 3 B) 1, 2, 4 C) 1, 4, 5 D) 1, 3, 4 E) 2, 3, 4 5. There is reciprocal regulation of glycolytic and gluconeogenic reactions interconverting fructose-6-phosphate and fructose-1,6-bisphosphate. Which one of the following statements about this regulation is not correct? A) Fructose-2,6-bisphosphate activates phosphofructokinase-1. B) Fructose-2,6-bisphosphate inhibits fructose-1,6-bisphosphatase. C) The fructose-1,6-bisphosphatase reaction is exergonic. D) The phosphofructokinase-1 reaction is endergonic. E) This regulation allows control of the direction of net metabolite flow through the pathway.

6. 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.

7. Conversion of 1 mol of acetyl-CoA to 2 mol of CO2 and CoA via the citric acid cycle results in the net production of: A) 1 mol of citrate.

B) 1 mol of FADH2. C) 1 mol of NADH. D) 1 mol of oxaloacetate. E) 7 mol of ATP.

On the next three pages are three questions dealing with glycolysis, the TCA cycle, and the electron transfer complexes of mitochondria. Each question is worth 20 points, and you must answer two of the three questions. If you answer all three I will give you the grade earned on the best two questions.

-2- Alternate question 1: 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 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

-3- Alternate question 2: 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- Alternate question 3: Diagram the flow of electrons from NADH or succinate all the way combining with oxygen to make water. Include all complexes, all electron carriers not located within a complex and any protons transported across the membrane.

A diagram like figure 19-16 of your text is a good start Complex I NADH + H+ 6NAD+ +6 Q + 2H QH2 4 Protons inside 64 protons outside Complex II 6 Succinate + Q Fumarate + QH2 Complex III 6 QH2 + 2 cytochrome cox Q + 2cytochrome cred + 2 protons outside 2 protons inside 6 2 protons outside Complex IV 6 2 cytochrome cred 2 cytochrome cox + 6 2 H inside + ½ O2 H2O 2 protons inside 6 2 protons outside

Second set of alternate questions. Each question is worth 15 points and again you must answer 2 of 3. Alternate set 2-question 1. Explain how ATP syntase works and why we don’t get an integral number of ATPs synthesized for each NADH Protons on the outside of the inner mitochondrial membrane are allowed to pass back to the inside of the membrane by passing through the a and c subunits of the FO complex. If the Fo complex has 8 c subunits as in yeast, then it takes 8 protons to make o this unit turn 360 , if it has 10 c subunits then it takes 10 protons to make the F0 o complex to a 360 turn. The turning of the FO subunit is coupled to the F1 complex by o the ã protein. A complete 360 of both the FO and F1 turn converts 3 ADP + Pi’s into 3 ATP. Thus it appears that in yeast 8 protons make 3 ATP. However to make each ATP we have to use a proton to transport inorganic phosphate into in mitochondrial matrix, so it takes 11 protons/3 ATP or 3.7 protons/ATP (NOT 3.5 which you only get if you use 9 c subunits!)

So in yeast, where 1 NADH pumps a total of 10 protons across the membrane 10 protons * (1 atp/3.7 protons) = 2.7 ATP/NADH yeast

If you do these calculations with 10 c subunits that are found in e coli you test 10+3 = 13 protons/3 ATP or 4.3 protons/ATP 10 protons * (1/4.3) =2.3 ATP/NADH

So we will go with a nice average of 2.5!

-5- Alternate set 2 question 2. Discuss the effects of insulin and glucagon on the glycolysis and gluoconeogenesis in as much detail as possible. (Ie. What specific enzymes are turned on and off or how is their activity is modulated)

This first part is also used inthe answer to the next question! Insulin 1 - effect on Phosphofructokinase 1 Insuline stimulates phosphoprotein phosphatase phorphoprotein phosphatase removes phosphate from the PFK-2/FBPase-2 complex activates PFK-2 deactivates FBPase-2

F26BP levels rise F26BP in turn stimulates PFK-1 activity rises and FBPase-1 activity falls and glycolysis is turned on and gluconeogenesis is slowed

Glucagon 1 - Effect on Fructose 1,6 bisphosphae phosphatase Glucogon stimulates the release of cAMP cAMP simulates cAMP dependent protein kinase cAMP protein kinase phosphorylates the PFK-2/FBPase-2 complex The phosphorylated PFK-2/FBPase-2 complex turns off the PFK-2 activity and increases the FBPase-2 activity levels of F26BP fall PFK-1 activity drops and FBPase-1 is stimulated Glycolysis is turned off and gluconeogenesis is turned on

Effect 2 Glucogon stimulates the release of cAMP cAMP simulates cAMP dependent protein kinase cAMP dependent protein kinase phosphorylates the isozyme of pyruvate kinase found in the liver the phosphorylated pyruvate kinase is inhibited and glycolysis is shut down

Effect 3 Finally back in chapter 14 we had a brief discussion of diabetes. In this discussion I mentioned that one of the effects of insulin, through a couple of intermediates is to make vesicles containing the GLUT4 transporter to fuse with cell membrane. As you get more transporters on the cell surface, you get more glucose in the cell, and glycolysis gets stimulated.

-6- Alternate set 2 question 3. Fructose 1,6 bisphosphate in an intermediate in both glycolysis and gluconeogenesis, Fructose 2,6-bisphosphate is an effector that regulates both glycolysis and gluconeogeneis. Discuss the role of Fructose 2,6-bisphosphate as a regulatory signal.

Fructose 2,6-bisphosphate is an allosteric effector of Phosphofructokinase-1(PKF-1) and Fructose 1,6-bisphosphatase (FBPase-1). Fructose 2,6-bisphosphate greatly increases the affinity of PKF-1 for Fructose 6-phosphate thus increasing the rate of glycolysis and greatly lowers the affinity of FBPase-1 for Fructose 1,6-bisphosphaste thus inhibiting gluconeogenesis. The levels of Fructose 2,6-bisphosphate are, in turn, regulated by an enzyme that has both PKF-2 and FBPase-2 activity located on a single bifunctional protein. The PFK-2 activity is turned on and the FBPase-2 turned off by de-phosphorylation of the enzyme by phosphoprotein phosphatse. Conversely the FBPase-2 activity is turned on and the PFK-2 is turned off by the phosphorylation of the enzyme by cAMP dependent protein kinase. These phosphorylation and dephosphorylation events are regulated by Insulin and glucagon as given in the previous question.

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