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DENF 1521 Biochemistry

Dental Biochemistry

Lesson 5.3 ATP Synthesis

5.3 The Synthesis of ATP

5.3A Lesson objectives

The objectives of this lesson are to understand:

1. The relationship between the structure of the mitochondrial ATP synthase and its function
2. The mechanism of ATP synthase
3. The role of the ATP/ADP translocase
4. The efficiency of oxidative phosphorylation for ATP production
5. Respiratory control of oxidative phosphorylation

5.3B ATP Synthase structure

In the previous lesson you learned how the transport of electrons down the mitochondrial respiratory chain generated a proton gradient across the inner mitochondrial membrane. Now this proton gradient will be used to generate ATP. Remember that the generation of the proton gradient and the synthesis of ATP are coupled but are separate processes. The enzyme responsible for the synthesis of ATP is ATP synthase also called ATPase.

The mitochondrial ATP synthase (ATPase) is a complex enzyme containing many subunits. Each subunit has a specific function and is composed of multiple polypeptide chains.

SUBUNITS	MW	SUBUNIT COMPOSITION; NUMBER	FUNCTION	LOCATION
F1	378,000		Synthesize ATP; & form catalytic site. Remaining 3 subunits form link (stalk) to F0	Matrix side
F0	66,000	4 subunits	Contains proton channel	Transmembrane
F1 inhibitor	10,000	Single polypeptide	Regulate proton flow and ATP synthesis	Stalk between F1 & F0
Oligomycin-sensitivity conferring protein (OSCP)	23,000	Single polypeptide	Regulate proton flow and ATP synthesis	Stalk between F1 & F0
Fc2 inhibitor	8,000	Single polypeptide	Regulate proton flow and ATP synthesis	Stalk between F1 & F0

The ATP synthase is oriented in the inner mitochondrial membrane such that the F₀ subunit is imbedded in the membrane. On the matrix side of the membrane is the F₁ inhibitor subunit (the stalk) and the F₁ subunit. Remember that the F₁ subunit contains the catalytic unit and therefore, ATP is synthesized on the matrix side of the mitochondrial membrane. Oligomycin is an antibiotic which binds to the mitochondrial ATP synthase and is a potent inhibitor of ATP synthesis and electron transport.

An excellent representation of ATP synthesis in the mitochondria is available in an animation at the following University of Connecticut site. Note the orientation and arrangement of subunits in the ATP synthase complex. Another illustration of these events can be found at the following site.

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

ATP synthase structure
Practice Exercise 1:	The F1 subunit of ATP synthase is responsible for No Response Movement of protons Synthesis of ATP Uncoupling the proton gradient Binding of oligomycin
Practice Exercise 2:	Which of the following parts of ATP synthase are found within the mitochondrial membrane? No Response F1 F0 Fc The oligomycin-sensitivity-conferring protein
Practice Exercise 3:	The actual synthesis of ATP takes place on the cytosolic side of the inner mitochondrial membrane. No Response True False
Practice Exercise 4:	The synthesis of ATP and electron transport are coupled under ordinary metabolic conditions. No Response True False

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

5.3C Mechanism of ATP synthesis

Dr. Paul D. Boyer, University of California, Los Angeles and Dr. John E. Walker, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom elucidated the enzymatic mechanism underlying the synthesis of ATP. They shared 1/2 of the 1997 Nobel prize in chemistry for this discovery. This mechanism is called the binding exchange mechanism of ATP synthesis. This model predicts that each catalytic site has a different conformation at any one point in time. As shown in the diagram below, the ATP synthase molecule contains 3 active sites. These 3 catalytic sites cycle through 3 different conformational states.

1. Open	Low substrate affinity
2. Tight	Active; High substrate affinity
3. Loose	Inactive; low substrate affinity

The implications of this model are that the

1. proton gradient drives the interconversion of the sites
2. proton gradient leads to the release of tightly bound ATP
3. actual synthesis of ATP from ADP and P_i occurs in the absence of the pH gradient

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

Mechanism of ATP synthesis
Practice Exercise 5:	What role does the proton gradient play in the synthesis of ATP from ADP and Pi by ATP synthase? No Response Required to catalyze addition of Pi to ADP Leads to release of ATP from ATP synthase Carries Pi into the mitochondrial matrix Required for ADP binding to ATP synthase
Practice Exercise 6:	For the binding-change mechanism of ATP synthesis to work, all catalytic sites must have the same conformational state. No Response True False

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

5.3D ATP/ADP Translocase

The mitochondrial ATP/ADP translocase is involved in the entry of ADP into the mitochondria. The translocase catalyzes the coupled entry of ADP to the exit of ATP from the mitochondria. Thus, ADP enters the mitochondria only if ATP exits. This coupled flow is essential for oxidative phosphorylation to continue. The translocase cycle is driven by the membrane potential. The proposeed mechanism is shown in the following diagram.

Note that ADP from the cytosol [ATP (cyt)] first binds to the translocate. After eversion of the translocase the ADP is now in the mitochondria [ADP (mito)]. The translocase then binds ATP from the mitochondria [ATP (mito)] and after another eversion the ATP the ATP is released into the cytosol. ATP exits the mitochondria 30-times faster than ADP enters. This means that as soon as ATP forms in the mitochondria it is transported to the cytosol by the translocase.

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

Mechanism of ATP synthesis
Practice Exercise 7:	The function of the ATP/ADP translocase is to No Response Catalyze the coupled entry of ADP and the exit of ATP from the mitochondria Catalyze the actual synthesis of ATP Establish a proton gradient Act as an uncoupler to inhibit ATP synthesis
Practice Exercise 8:	ATP and ADP can freely enter or exit the mitochondria. No Response True False

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

5.3E ATP yield

The following table summarizes the steps involved in the production of ATP by the cell. The following pathways produce ATP or NADH which is eventually leads to the formation of ATP.

Reaction	ATP/glucose
Glycolysis in cytosol
Phosphorylation of glucose	-1
Phosphorylation of fructose 6-phosphate	-1
Dephosphorylation of 2 molecules 1,3-bisphosphoglycerate	+2
Dephosphorylation of 2 molecules phosphoenolpyruvate	+2
Oxidation of 2 molecules glyceraldehyde 3-phosphate yields 2 NADH
Glycolysis
Conversion of 2 molecules pyruvate to acetyl CoA in mitochondria yields 2 NADH
Citric acid cycle in mitochondria
2 GTP formed from 2 molecules succinyl CoA	+2
Oxidation of 2 molecules each of isocitrate, -ketoglutarate, and malate yields 6 NADH
Oxidation of 2 molecules of succinate yields 2 FADH2
Oxidative phosphorylation in mitochondria
2 molecules NADH from glycolysis assuming glycerol phosphate shuttle* (or malate/aspartate shuttle*)	+3 (+5)
2 molecules NADH from conversion of pyruvate to acetyl CoA	+5
6 molecules NADH from citric acid cycle	+15
2 molecules FADH2 from citric acid cycle	+3
Net yield/glucose	+30 (+32)

*see next lesson for a further explanation of the different shuttle systems for getting NADH from the cytosol to the mitochondria.

Be aware that the "traditional" estimate of the number of ATP's/glucose is 36 (2 ATP/NADH). The currently accepted value is 1.5 ATP/NADH and this number is used in the above table. For more details see may see Hinkle, et al.

5.3F Efficiency of biologic oxidation of glucose

Using the yield of 30 ATP/glucose molecule oxidized by a conbination of glycolysis, the citric acid cycle, and oxidative phosphorylation results in a calculated G of 219 kcal/mol (30 ATP x -7.3 kcal/mol). The "burning" of glucose to directly yield 6 CO₂ and 6 H₂O has a G of 686 kcal/mol. This gives an overall efficiency of 32% (219/686 = 0.32). Note that 26 of the 30 ATP generated come from oxidative phosphorylation.

A very good drawing indicating the relationships between the different processes taking part in the mitochondria can be found at this University of Virginia site.

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

ATP yield and efficiency
Practice Exercise 9:	The biologic oxidation of glucose is much MORE efficient than a chemical oxidation. No Response True False
Practice Exercise 10:	Most of the ATP produced by the cell occurs in the mitochondria. No Response True False

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

5.3G Respiratory Control

Oxidative phosphorylation is regulated by the level of ADP. Electrons can only be trasferred to O₂ by the electron transport chain if ADP is phosphorylated to form ATP. Therefore, the level of ADP is the most important factor regulating oxidative phosphorylation. The [ADP] will increase as ATP is used by the cell so oxidative phosphorylation is coupled to the use of ATP. As a consequence, electrons do not flow from fuel molecules such as glucose to O₂ unless there is a need for ATP to be synthesized. This relationship (amount of O₂ consumed in relationship to the ADP levels) is shown in the following graph. Note that, with time, the ADP is comsumed and the rate of O₂ consumtion decreases.

5.3H Proton gradients

The proton gradient generated in a cell is not only used for the synthesis of ATP. A proton gradient is an interconvertible form of free energy and therefore, can be used to drive several types of biological processes. Among these other uses are:

• Heat production
• NADPH synthesis
• Flagellar rotation
• Active transport
• Electron potential

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

Respiratory control
Practice Exercise 11:	The most important factor directly controlling the rate of oxidative phosphorylation is the cell's level of No Response Glucose ADP ATP NAD+
Practice Exercise 12:	The amount of oxygen consumed by the cell decreases as the level of ADP decreases. No Response True False
Practice Exercise 13:	Proton gradients can only be used for the production of ATP No Response True False

Dental Biochemistry

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Lesson 5.3 ATP Synthesis

5.3I Summary

After completing this lesson you should understand the details of the formation of ATP by the mitochondrial ATP Synthase.

1. The mitochondrial ATP synthase is a multisubunit complex with a specific oreientation in the inner mitochondrial membrane
2. The binding exchange mechanism of ATP synthesis requires conformational changes
3. The actual synthesis of ATP can occur without the pH gradient
4. The pH gradient allows the release of ATP
5. The entry of ADP into the mitochondria. is coupled to the exit of ATP
6. The level of ADP is the most important regulatory control of oxidative phosphorylation

Final Instructions

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