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ATP Synthase ATP Synthase Lecture # 14 Lecture # 14

ATP Synthase

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ATP Synthase. Lecture # 14. What is ATP Synthase?. ATP (adenosine triphosphate) – the molecule of energy for cells, drives many biochemical reactions like muscle contraction, DNA and protein synthesis, etc ATP Synthase- enzyme that catalyses ATP synthesis and hydrolysis. - PowerPoint PPT Presentation

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Page 1: ATP Synthase

ATP SynthaseATP SynthaseLecture # 14Lecture # 14

Page 2: ATP Synthase

What is ATP Synthase?What is ATP Synthase? ATP (adenosine triphosphate) – the molecule of ATP (adenosine triphosphate) – the molecule of

energy for cells, drives many biochemical energy for cells, drives many biochemical reactions like muscle contraction, DNA and reactions like muscle contraction, DNA and protein synthesis, etcprotein synthesis, etc

ATP Synthase- enzyme that catalyses ATP ATP Synthase- enzyme that catalyses ATP synthesis and hydrolysissynthesis and hydrolysis

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/ATP.html

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Where is it found?Where is it found?

A membrane enzyme:A membrane enzyme: Found in:Found in:

bacterial plasma bacterial plasma membranesmembranes

The thylacoid membrane in The thylacoid membrane in chloroplastschloroplasts

The inner mitochondrial The inner mitochondrial membrane of eukaryotic membrane of eukaryotic cellscells

http://www.biologie.uni-osnabrueck.de/biophysik/Feniouk/images/Rb_capsulatus.jpg

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Parts of ATP SynthaseParts of ATP Synthase

Consists of two domains FConsists of two domains F00 and F and F11

http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Key_Resources/Metabolic_Pathways/ATP_Synthase.html

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The DomainsThe Domains Hydrophobic FHydrophobic F0 0 domain sits in the membrane - domain sits in the membrane -

performs proton translocation performs proton translocation Hydrophillic FHydrophillic F11 portion protrudes from portion protrudes from

membrane - performs ATP membrane - performs ATP synthesis/hydrolysis synthesis/hydrolysis 3 alternating alpha 3 alternating alpha and beta subunits and beta subunits

http://nobelprize.org/chemistry/laureates/1997/illpres/boyer-walker.html

Page 6: ATP Synthase

What does it do?What does it do?

It makes ATP from ADP and inorganic It makes ATP from ADP and inorganic phosphate (Pphosphate (Pii) )

ADP + PADP + Pi i + ATP+ ATP

The overall equation is: The overall equation is:

        ADPADP3-3- + HPO + HPO442-2- + H + H++ + nH + nH++

outside membrane(+ charge)outside membrane(+ charge)     

    ATPATP4-4- + H + H22O + nHO + nH++inside membrane (- charge)inside membrane (- charge)

energy from protons diffusing energy from protons diffusing across membrane with the gradientacross membrane with the gradient

Page 7: ATP Synthase

How does it function?How does it function?

First a proton gradient is First a proton gradient is establishedestablished Protons collect on one side of Protons collect on one side of

the membranethe membrane

Then the protons flow Then the protons flow through a channel in the through a channel in the enzyme causing the protein enzyme causing the protein subunits to rotatesubunits to rotate

http://www.sp.uconn.edu/~terry/images/anim/ATPmito.html

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The “stalk” rotates in 120° The “stalk” rotates in 120° incrementsincrements

causes the units in the Fcauses the units in the F1 1

domain to contract and domain to contract and expandexpand

The structural changes The structural changes facilitate the binding of facilitate the binding of ADP and PADP and Pi i to make ATPto make ATP

Each subunit goes through 3 Each subunit goes through 3 stagesstages Open State – releases any Open State – releases any

ATPATP Loose State – ADP and PLoose State – ADP and Pii

molecules enter the subunitmolecules enter the subunit Tight State – the subunit Tight State – the subunit

contracts to bind molecules contracts to bind molecules and make ATP and make ATP

Page 9: ATP Synthase

AnimationsAnimations

http://www.stolaf.edu/people/giannini/flashanimat/metabolism/atpsyn1.swf

Protons cross membrane through the ATP Protons cross membrane through the ATP synthase enzymesynthase enzyme

http://www.stolaf.edu/people/giannini/flashanimat/metabolism/atpsyn2.swf

Rotary motion of ATP synthase powers Rotary motion of ATP synthase powers the synthesis of ATP the synthesis of ATP

Page 10: ATP Synthase

Interesting FactsInteresting Facts Contains 22722 atoms Contains 22722 atoms 23211 bonds connected as 2987 23211 bonds connected as 2987

amino acid groupsamino acid groups 120 helix units and 94 sheet units 120 helix units and 94 sheet units Generates over 100 kg of ATP Generates over 100 kg of ATP

daily (in humans) daily (in humans) One of the oldest enzymes-One of the oldest enzymes-

appeared earlier then appeared earlier then photosynthetic or respiratory photosynthetic or respiratory enzymesenzymes

Smallest rotary machine known Smallest rotary machine known

Picture http://webct.uga.edu/public/FRES1010CG/BCMB401098ATPSyn2.gif/Picture http://webct.uga.edu/public/FRES1010CG/BCMB401098ATPSyn2.gif/

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Our ModelOur Model

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Enzyme Activity and Enzyme Activity and unitsunits

Page 13: ATP Synthase

Enzyme unitsEnzyme units

The enzyme unit (U) is a unit for the amount of a particular enzyme used for its activity.

“One U is the amount of the enzyme that catalyzes 1 micro mole of substrate per minute”.

The conditions also have to be specified: one usually takes as STP

Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.

Page 14: ATP Synthase

Enzyme ActivityEnzyme Activity

Unit of enzyme activity:Enzyme activity = moles of substrate converted per unit time = rate × reaction volumeUsed to measure total units of activity in a given volume of solution.Specific activity:It is the amount of product formed by an enzyme in a given amount of time under given conditions per mg of total protein. = rate × reaction volume / mass of total protein.

Molecular activity: Used to compare activities of different enzymes.

Page 15: ATP Synthase

Enzyme ActivityEnzyme Activity

Classical units:

Unit of enzyme activity:mol substrate transformed/min = unit

Specific activity:mol substrate/min-mg E = unit/mg E

Molecular activity:mol substrate/min- mol E = units/mol E

Page 16: ATP Synthase

Enzyme ActivityEnzyme Activity

New international units:

Unit of enzyme activity:mol substrate/sec = katal

Specific activity:mol substrate/sec-kg E = katal/kg E

Molecular activity:mol substrate/sec-mol E = katal/mol E

Page 17: ATP Synthase

Example 1Example 1

The rate of an enzyme catalyzed reaction is 35 μmol/min at [S] = 10-4 M, (KM = 2 x 10-5).Calculate the velocity at [S] = 2 x 10-6 M.

Work the problem.

Page 18: ATP Synthase

Example 1 AnswerExample 1 AnswerThe rate of an enzyme catalyzed reaction is 35 μmol/min at [S] = 10-4 M, (KM = 2 x 10-5).Calculate the velocity at [S] = 2 x 10-6 M.

First calculate VM using the Michaelis-Menton eqn: VM [S] VM (10-4) VM (10-4)v = -----------, so: 35 = ------------------ = -------------- KM + [S] 2 x 10-5 + 10-4 1.2 x 10-4

VM = 1.2(35) = 42 mol/min; then calculate v:

42 (2 x 10-6) 84 x 10-6 v = ------------------------ = ------------ = 3.8 mol/min

2 x 10-5 + 2 x 10-6 22 x 10-6

Page 19: ATP Synthase

Example 2Example 2

An enzyme (1.84 μgm, MW = 36800) catalyzes a reaction in presence of excess substrate at a rate of 4.2 μmol substrate/min. What is the TON in min-1 ? What is the TON in sec-1 ?

Work the problem.

Page 20: ATP Synthase

Example 2 AnswerExample 2 Answer

An enzyme (1.84 μgm, MW = 36800) catalyzes a reaction in presence of excess substrate at a rate of 4.2 μmol substrate/min. What is the TON ?

1.84 μgm μ mol E = ------------------------- = 5 x 10-5 μmol E

36800 μgm/ μmol

4.2 μmol/min TON = ------------------ = 84000 min-1

5 x 10-5 μmol

Page 21: ATP Synthase

Example 2 AnswerExample 2 Answer

What is the value of this TON (84000 min-1) in units of sec-1 ?

84000 min-1 1 sec-1

TON E = ------------------ x ---------- = 1400 sec-1

60 min-1

Page 22: ATP Synthase

Example 3Example 3

Ten micrograms of carbonic anhydrase (MW = 30000) in the presence of excess substrate exhibits a reaction rate of 6.82 x 103 μmol/min.At [S] = 0.012 M the rate is 3.41 x 103 μmol/min.

a. What is Vm ? b. What is KM ?

Work these.

Page 23: ATP Synthase

Example 3Example 3

a. The rate in presence of excess substrate is Vmaxso:

Vmax = 6.82 x 103 μmol/min.

b. At [S] = 0.012 M the rate is 3.41 x 103 μmol/min which is ½ Vmax so:

KM = 0.012 M. This may also be determined using the

Michaelis-Menton equation.