ATP SynthaseLecture # 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, etcATP Synthase- enzyme that catalyses ATP synthesis and hydrolysis
Where is it found?A membrane enzyme:Found in: bacterial plasma membranesThe thylacoid membrane in chloroplastsThe inner mitochondrial membrane of eukaryotic cellshttp://www.biologie.uni-osnabrueck.de/biophysik/Feniouk/images/Rb_capsulatus.jpg
Parts of ATP SynthaseConsists of two domains F0 and F1
The Domains Hydrophobic F0 domain sits in the membrane - performs proton translocation Hydrophillic F1 portion protrudes from membrane - performs ATP synthesis/hydrolysis 3 alternating alpha and beta subunits http://nobelprize.org/chemistry/laureates/1997/illpres/boyer-walker.html
What does it do?It makes ATP from ADP and inorganic phosphate (Pi) ADP + Pi + ATPThe overall equation is: ADP3- + HPO42- + H+ + nH+outside membrane(+ charge) ATP4- + H2O + nH+inside membrane (- charge) energy from protons diffusing across membrane with the gradient
How does it function?First a proton gradient is establishedProtons collect on one side of the membraneThen the protons flow through a channel in the enzyme causing the protein subunits to rotate
The stalk rotates in 120 increments causes the units in the F1 domain to contract and expandThe structural changes facilitate the binding of ADP and Pi to make ATPEach subunit goes through 3 stagesOpen State releases any ATPLoose State ADP and Pi molecules enter the subunitTight State the subunit contracts to bind molecules and make ATP
Animationshttp://www.stolaf.edu/people/giannini/flashanimat/metabolism/atpsyn1.swf Protons cross membrane through the ATP synthase enzymehttp://www.stolaf.edu/people/giannini/flashanimat/metabolism/atpsyn2.swf Rotary motion of ATP synthase powers the synthesis of ATP
Interesting FactsContains 22722 atoms 23211 bonds connected as 2987 amino acid groups120 helix units and 94 sheet units Generates over 100 kg of ATP daily (in humans) One of the oldest enzymes-appeared earlier then photosynthetic or respiratory enzymesSmallest rotary machine known
Enzyme Activity and 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 STPAmounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.
Enzyme ActivityUnit 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.
Enzyme ActivityClassical units:
Unit of enzyme activity:mmol substrate transformed/min = unit
Specific activity:mmol substrate/min-mg E = unit/mg E
Molecular activity:mmol substrate/min- mmol E = units/mmol E
Enzyme ActivityNew 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
Example 1The rate of an enzyme catalyzed reaction is 35 mol/min at [S] = 10-4 M, (KM = 2 x 10-5).Calculate the velocity at [S] = 2 x 10-6 M.
Work the problem.
Example 1 AnswerThe rate of an enzyme catalyzed reaction is 35 mol/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 mmol/min;then calculate v:
42 (2 x 10-6) 84 x 10-6 v = ------------------------ = ------------ = 3.8 mmol/min 2 x 10-5 + 2 x 10-6 22 x 10-6
Example 2An enzyme (1.84 gm, MW = 36800) catalyzes a reaction in presence of excess substrate at a rate of 4.2 mol substrate/min. What is the TON in min-1 ? What is the TON in sec-1 ?
Work the problem.
Example 2 AnswerAn enzyme (1.84 gm, MW = 36800) catalyzes a reaction in presence of excess substrate at a rate of 4.2 mol substrate/min. What is the TON ?
1.84 gm mol E = ------------------------- = 5 x 10-5 mol E 36800 gm/ mol
4.2 mol/min TON = ------------------ = 84000 min-1 5 x 10-5 mol
Example 2 AnswerWhat is the value of this TON (84000 min-1) in units of sec-1 ?
84000 min-1 1 sec-1TON E = ------------------ x ---------- = 1400 sec-1 60 min-1
Example 3Ten micrograms of carbonic anhydrase (MW = 30000) in the presence of excess substrate exhibits a reaction rate of 6.82 x 103 mol/min.At [S] = 0.012 M the rate is 3.41 x 103 mol/min.
a. What is Vm ? b. What is KM ?
Example 3The rate in presence of excess substrate is Vmaxso: Vmax = 6.82 x 103 mol/min.
b. At [S] = 0.012 M the rate is 3.41 x 103 mol/min which is Vmax so: KM = 0.012 M. This may also be determined using the Michaelis-Menton equation.