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DENF 1521 Biochemistry

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Oxidative Phosphorylation

Lesson 5.3
The Synthesis of ATP

Instructions
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  2. Then study Lesson 5.3 at your own pace. When Practice Exercises appear, click the appropriate button to choose your answer. Then press the "Get Feedback..." button to find out how you did. Continue to try again if you miss.
  3. After studying Lesson 5.3, and responding to all practice exercises, follow instructions at the end to submit your responses for Lesson 5.3 participation credit.
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DB Bullet 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 action 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 lesson 5.2 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 Oligomycin is antibiotic that
blocks utilization of proton gradient 		Stalk between F1 & F0
Fc2 inhibitor 8,000 Single polypeptide   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 Brush
DB Bullet 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

 

 

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DB Bullet 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 Pi occurs in the absence of the pH gradient

 

 

 

 

 

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DB Bullet 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


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DB Bullet 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 proposed 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 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 Brush
DB Bullet 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


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DB Bullet 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 Hinkle and Yu

5.3F Efficiency of biologic oxidation of glucose

Using the yield of 30 ATP/glucose molecule oxidized by a combination 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 Brush
DB Bullet Lesson 5.3 ATP Synthesis
ATP yield and efficiency
Practice
Exercise 9: 		The biologic oxidation of glucose is much MORE efficient than its chemical oxidation.

No Response
True
False


Practice
Exercise 10: 		Most of the ATP produced by the cell is synthesized in the mitochondria.

No Response
True
False


Dental Biochemistry Brush
DB Bullet Lesson 5.3 ATP Synthesis

5.3G Respiratory Control

Oxidative phosphorylation is regulated by the level of ADP. Electrons can only be transferred 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 consumed and the rate of O2 consumption 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:

Dental Biochemistry Brush
DB Bullet 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 Brush
DB Bullet 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 orientation in the inner mitochondrial membrane
  2. The binding exchange mechanism of ATP synthesis requires conformational changes in the synthase complex
  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

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End Lesson 5.3
Synthesis of ATP



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