Answers 503 Lecture Discussion Week 6 02/28/14 & 03/03/14

*Remember: Good ways to learn and understand reactions rather than simply memorizing will be presented. Emphasis will be placed on the important principles that will help you learn structures and predict other substrates and products in reactions. Dr. Hull will give you one structure (either substrate or product) for a given reaction in glycolysis and the TCA cycle , and you will be expected to complete the reaction with the other structure. You will have to understand what is going on in each reaction rather than just memorizing it.


1. a & b. Using structural formula's, give two mitochondrial reactions in which a decarboxylation is coupled to an oxidation and a high energy thioester bond is formed. Name the enzymes and all substrates, cofactors and products.

c. Name the three cofactors used catalytically in the oxidative decarboxylation of both pyruvate and α-ketoglutarate (both are alpha keto acids).

TPP, lipoamide (sometimes called lipoic acid), and FAD

d.Briefly indicate their role in this reaction.

TPP: involved in catalyzing the decarboxylation of pyruvate

lipoamide: involved in the transfer of the acetyl group to the HS-CoA

FAD: involved in regeneration of the oxidized form of lipoamide


e. Using structural formula's draw: A TCA cycle reaction in which an alcohol is oxidized to a ketone but the reaction is irreversible.


f. Using structural formula's draw: A TCA cycle reaction in which the hydrolysis of a thioester is coupled to the formation of a carbon-carbon bond.


2. Identify the following reactions taking place inside the mitochondria by giving the enzyme name or naming both substrate and product(i.e. fumarate to malate). More than one reaction may be possible.


a. An oxidative decarboxylation of an α-keto acid
:
α-ketoglutarate to succinyl-CoA catalyzed by α-ketogluterate dehydrogenase in the TCA cycle.
Also pyruvate to acetyl-CoA catalyzed by pyruvate dehydrogenase directly following glycolysis.

b. A substrate level phosphorylation to make a nucleotide triphosphate equivalent to an ATP:
succinyl-CoA + GDP + Pi -> succinate + GTP

c.Three biologically irreversible steps of the TCA cycle:
Citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and succinate dehydrogenase reactions.

d. A reaction that utilizes a FAD in a reaction linked directly to electron transport.:
succinate to fumarate catalyzed be succinate dehydrogenase in the TCA cycle

3. Oxidative Phosphorylation is the process by which the energy released during electron transport is coupled to ATP synthesis.
a. Indicate the key landmarks in the electron transport chain.


NADH ---> NADH Dehydrogenase (Complex I)--> CoQ ---> Ubiquinol Cyt C Reductase (Complex III)--> cyt C----> Cytochrome Oxidase (Complex IV)-->Oxygen
Succinate Dehydrogenase (Complex II, located after NADH Dehydrogenase) accepts from FADH2 & passes its electrons directly onto CoQ

b. Cyanide binds to the cytochrome oxidase of complex IV and prevents oxygen reduction to water. What effect would you expect this to have on the rate of electron transport? What about the rate of the ATP synthase reaction?


The rate of electron transport would slow or stop, because cyanide is acting as an inhibitor of cytochrome oxidase and therfore the electron transport chain (ETC). If the flow of electrons cannot be maintained, the protons can no longer be pumped out of the mitochondrial matrix. The proton gradient will not be maintained.

4. How many net ATP's are produced when the pyruvate is oxidized to CO2 and H2O in the mitochondria via the pyruvate dehydrogenase reaction, TCA cycle, electron transport, substrate level and oxidative phosphorylation? Show your work.

2 pyruvate are produced from one glucose. The two pyruvates will go through the pyruvate dehydrogenase reaction and the TCA cycle reactions.

pyruvate--> acetylCoA 1 NADH * 3 ATP/NADH (* 2 pyruvate = 6 ATP)



isocitrate --> α-ketoglutarate 1 NADH * 3 ATP/NADH (* 2 pyruvate = 6 ATP)



α-ketoglutarate --> succinyl CoA 1 NADH * 3 ATP/NADH (* 2 pyruvate = 6 ATP)


succinyl CoA ---> succinate 1 GTP = 1 ATP equivalent (* 2 pyruvate = 2 ATP equivalents)
( because of this reaction: GDP + ATP <-> GTP + ADP )

succinate --> fumarate 1 FADH2 * 2ATP/ FADH2 (* 2 pyruvate = 4 ATP)


malate --> oxaloacetate 1 NADH * 3 ATP/NADH (* 2 pyruvate = 6 ATP)


Total yield of ATP: 15 ATP for 1 pyruvate or 30 ATP for 2 pyruvates

5. A biotechnology firm hires you to run some tests on mammalian mitochondrial function. You prepare mitochondria and incubate them in abundant Pi, ADP, and O2.

A. Using malate as an energy source, you demonstrate that your mitochondrial preparation is fully proficient in the process of electron transport and oxidative phosphorylation, because __3____ ATP(# of ATP) are made per malate molecule oxidized. Note that in this test tube experiment malate is oxidized to oxaloacetate which then accumulates as a product.



B. You further test the response of the mitochondria to various treatments in the presence of malate. Indicate whether the treatments will increase or decrease the rate of electron transport compared to the condition described in part A.

___decrease___ 1. O2 is omitted, but other components are kept the same as in part A.

___decrease___ 2. Rotenone, an inhibitor of NADH dehydrogenase, is included with the components added in part A.

___increase___ 3. Dinitrophenol, an uncoupler, is included with the components added in part A.

__ decreased___ 4. Rotenone, an inhibitor of NADH dehydrogenase, is included with the components added in part A + succinate. (NOTE:succinate will come into the ETC at Succinate dehydrogenase, so you will be able to make 2 ATP...which is less than the 3 ATP made from NADH)

 

Glycogen Metabolism will be discussed and reviewed in discussion (or will be continued until next week).

6. In an exercising muscle, muscle glycogen is broken down and the carbons used to run glycolysis. Show the pathway in words to pyruvate.

Glycogen (glucosen) + Pi->Glucose-1-Phosphate+Glycogen (glucosen-1)

Glucose-1-Phosphate-> Glucose-6-Phosphate -> Fructose-6-Phosphate-> through glycolysis to pyruvate.

The first reaction is catalyzed by glycogen phosphorylase (phosphorylated form).

There will be 2 pyruvates made per glucose-6-phosphate.

7. In muscle after eating, muscle glycogen will be made. Show the pathway in words from blood glucose to muscle glycogen.

Blood Glucose transported into the muscle cell by glut-4.

Muscle glucose + ATP-> ADP + Glucose-6-Phosphate by the hexokinase reaction the an isomerase reaction Glucose-6-Phosphate-> Glucose-1-Phosphate then:

Glucose-1-phosphate + UTP -> UDP-Glucose + PPi

UDP-glucose + Glycogen (glucosen) -> Glycogen (glucosen+1) + UDP This reaction is catalyzed by Glycogen Synthase (dephosphorylated form)