ATPases; P-type, V-type & F-type, how does H + power V & F-type 2015

Transcription

1 ATPases; P-type, V-type & F-type, how does H + power V & F-type 2015

2 1 /2 O 2 +NADH 2 H 2 O + NAD + ΔE O =0.82-(-0.32)= 1.14V ΔG O = -218KJ/mol The oxidation of 1mol of NADH is associated with the release of 218kJ of free energy The formation of ATP from ADP + Pi ATP requires 30.5 kj/mol free energy input

3 The free energy necessary to generate ATP is extracted from the oxidation of NADH & FADH 2 by the ETS, a series of 4 complexes thru which e - pass from lower to higher standard reduction potentials and the release of free energy.

4 The changes in std. reduction potential of an e - pr. as it successively transverses complexes I, III, & IV, correspond at each stage to sufficient free energy to power the synthesis of an ATP.! Complex I ΔE oʹ = 0.36V ΔG oʹ = kj/mol! Complex III ΔE oʹ = 0.19 V ΔG oʹ = kj/mol! Complex IV ΔE oʹ = 0.58 V ΔG oʹ = kj/mol!

5 Complex II catalyzes the oxidation of FADH 2 by CoQ,! FADH 2 +CoQ FAD + CoQH 2! ΔE Oʹ V ΔG O -16.4kJ/mol! This redox rxn. does not release sufficient free energy to synthesize an ATP, it functions only to bring e - from FADH 2 into the ETS, therefore e- entering the ETS chain will have enough free energy to generate 2 ATP.!

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7 Putative dimeric structure of the (Na + K + ) ATPase indicating its orientation in the plasma membrane.! Scheme of the membrane topology of the α - and β- isoforms of the Na + -K + -ATPase. Sequences of rat α 1- and β 1- isoforms are shown. Residues are colored to indicate the amino acid homology among the different α -isoforms (1, 2, 3, and 4) or β -isoforms (1, 2, and 3).!

8 Mechanistic scheme for the active transport of Na + and K + by! (Na + K + ) ATPase.! The free energy of ATP hydrolysis powers the endergonic transport of Na + & K + against the electrochemical gradient. This pump works by confromational change due to Aspartyl phosphorylation & dephosphorylation. The enzyme appears to have only one binding site that changes confromation with the phosphorylation of the Aspartyl site. This phosphorylation changes both the proteins orientation and specificity.!

9 Kinetic mechanism of Ca 2+ ATPase. These pump Ca out of the cytosol and into either the lumen of E.R or out of the cell.! Structure of the Ca 2+ ATPase from rabbit muscle sarcoplasmic reticulum..also present in plasma membr.

10 Although the immediate energy for glucose transport Is the Na + gradient, the gradient is due to the Na + -K + P- type ATPase & the hydrolysis ATP to maintain the gradient inside the brush border cells. Sodium-dependent glucose cotransporters (SGLT) are a family of glucose transporter found in the intestinal mucosa (enterocytes) of the small intestine (SGLT1) and the proximal tubule of the nephron (SGLT2 in PCT and SGLT1 in PST). They contribute to renal glucose reabsorption. In the kidneys, 100% of the filtered glucose in the glomerulus has to be reabsorbed along the nephron (98% in PCT, via SGLT2). In case of too high plasma glucose concentration (hyperglycemia), glucose is excreted in urine (glucosuria); because SGLT are saturated with the filtered monosaccharide. Glucose is never secreted by the nephron.

11 How is ATP generated in the mitochondria by the! F type H + ATP synthase?! Can ATP synthesis be uncoupled from the ETS?! Can the F type H + ATP synthase be reversed and hydrolyse ATP and pumping H +?! Are there structural similarities between the V-type H + ATPase and the F type H + ATP synthase?!

12 The uncouplers act to separate the ETS from Ox- Phos.! This was the indictation that there where 2 pathways connected by H + gradient (pmf).!

13 ATP synthesis is catalysed by the proton translocating ATP Synthase (Complex V) is driven by the ETS. But the ATPase Is physically distinct from Complexes I-IV. The free energy released by the ETS is conserved for the ATPase to utilize. The Chemiosmotic Hypothesis states that the H + pumping by the complexes produces a H + gradient and the electrochemical potential of this gradient is harnessed to synthesize ATP.

14 ETS causes complexes to transport protons across the inner mito membrane from matrix, from lower lower [H + ] & negative electrical potential to the intermembr space a region of high [H + ] and positive electrical potential. The ph of the matrix is 0.75 unit higher than the intermembr space. The free energy sequestered by the resulting electrochemical gradient is called the pmf (analogous to emf) drive ATP synthesis. ΔG for proton transport is 21.5kJ/mol. Approx. 3 protons are need to generate an ATP.

15 These mitochondrial inner-membrane transporters must work in unison to generate ATP and to deliver it to where it is necessary for cellular work. The stoichometry of ATP synthesis and H + flux is 3 H + per/atp + 1H + /PO 4.

16 Mitochondrial ATP Synthase E. coli ATP Synthase The free energy of the electrochemical proton gradient across the mitochondrial inner membrane is harnessed in the synthesis of ATP by the proton-translocating ATP synthase also called the F 1 F 0 ATPase, Complex V and the F- type H + -ATPase. It is a multi-subunited transmembrane protien. It has two major subunits comprised of 8 to 13 subunits each.

17 F 0 is a water soluble transmembr. protein composed of as many as 8 subunits (in E.coli it is 13). It contains the proton translocating channel. F 1 is a water soluble peripheralprotein that can be easily dissociated from the F 0 by urea treatment.!

18 F1 is an α 3 β 3 γδε nonamer in which! The β subunit contains the catalytic site of ATP synthesis and the δ is required to bind F 1 to F 0.!

19 F 0 is composed of 13 transmembranous subunits that forms a ring. It also contains one copy of 3 other proteins including the OSCP(δ protein in E.coli).

20 F 1 has 3 interacting catalytic protomers, each in a different conformational state. The free energy released on proton translocation is harnessed to interconvert these 3 states. ATP is synthesized in the T state and released in the O. How is the free energy of proton translocation coupled to coupled to ATP synthesis? The binding changes are driven by the rotation of the catalytic assembly α 3 β 3 with respect to the other portions of the F 1 F 0 ATPase. The γ subunit act as a rotating camshaft inside the α 3 β 3. It links the proton driven rotation to the conformational changes of the catalytic site of F 1.

21 ATP hydrolysis and synthesis occurs on 3 catalytic sites in the F 1 sector,peripheral to the membrane. Proton transport occurs through the membrane embedded F o. Direct evidence has been found for ATP hydrolysis driven rotation of a subset of, sub-units called the rotor, energy transmission from F 1 to F o and up-hill proton transport are functions of rotation. Hydrolysis and synthesis then depend on the direction of rotation.

22 V-ATPase, a true H + pump. V-ATPase is a highly conserved evolutionarily ancient enzyme with remarkably diverse functions in eukaryotic organisms.v-atpases acidify a wide array of intracellular organelles and pump protons across the plasma membranes of numerous cell types. V-ATPases couple the energy of ATP hydrolysis to proton transport across intracellular and plasma membranes of eukaryotic cells.

23 The yeast V-ATPase is the best characterized. There are at least 13 subunits identified to form a functional complex, which consists of two domains. The subunits belong to either the V o domain or the V 1 domain. The V 1 includes 8 subunits, A-H, with three copies of the catalytic A and B subunits, three copies of the stator subunits E and G, and one copy of the regulatory C and H subunits. In addition, the V 1 domain also contains the subunits D and F, which form a central rotor axel. The V 1 domain contains tissue-specific subunit isoforms including B, C, E, and G. The V o domain contains 6 different subunits, a, d, c, c', c", and e, with the stoichiometry of the c ring still a matter of debate with a decamer being postulated. The mammalian V o domain contains tissue-specific isoforms for subunits a and d, while yeast V-ATPase contains two organelle-specific subunit isoforms of a, Vph1p, and Stv1p. The V 1 domain is responsible for ATP hydrolysis, whereas the V o domain is responsible for proton translocation. ATP hydrolysis at the catalytic nucleotide binding sites on subunit A drives rotation of a central stalk composed of subunits D and F, which in turn drives rotation of a barrel of c subunits relative to the a subunit. These structures have revealed that the V-ATPase has a 3-stator network, linked by a collar of density formed by the C, H, and a subunits, which, while dividing the V 1 and V 0 domains, make no interactions with the central rotor axle formed by the F, D, and d subunits. Rotation of this central rotor axle caused by the hydrolysis of ATP within the catalytic AB domains results in the movement of the barrel of c subunits past the a subunit, which drives proton transport across the membrane. A stoichiomety of two protons translocated for each ATP hydrolyzed has been proposed.

24 In vivo regulation of V-ATPase activity is accomplished by reversible dissociation of the V 1 domain from the V o domain. After initial assembly, V-ATPases can reversibly disassemble into free V o and V 1 domains after a 2- to 5- minute deprivation of glucose. Reversible disassembly may be a general mechanism of regulating V- ATPase activity. Reassembly is proposed to be aided by a complex termed RAVE (regulator of H+- ATPase of vacuolar and endosomal membranes). Dissasembly and reassembly of V-ATPases does not require new protein synthesis but does need an intact microtubular network.

25 Evolution of the F1Fo ATPase The evolution of ATP synthase is thought to be an example of modular evolution during which two functionally independent subunits became associated and gained new functionality. This association appears to have occurred early in evolutionary history, because essentially the same structure and activity of ATP synthase enzymes are present in all kingdoms of life. The F-ATP synthase displays high functional and mechanistic similarity to the V-ATPase. However, whereas the F-ATP synthase generates ATP by utilising a proton gradient, the V-ATPase generates a proton gradient at the expense of ATP, generating ph values of as low as 1. The F 1 domain also shows significant similarity to hexameric DNA helicases, and the F O domain shows some similarity to H + -powered flagellar motor complexes. The α 3 β 3 hexamer of the F 1 domain shows significant structural similarity to hexameric DNA helicases; both form a ring with 3-fold rotational symmetry with a central pore. Both have roles dependent on the relative rotation of a macromolecule within the pore; the DNA helicases use the helical shape of DNA to drive their motion along the DNA molecule and to detect supercoiling, whereas the α 3 β 3 hexamer uses the conformational changes through the rotation of the γ subunit to drive an enzymatic reaction. The H + motor of the F O particle shows great functional similarity to the H + motors seen in flagellar motors. Both feature a ring of many small alpha-helical proteins that rotate relative to nearby stationary proteins, using a H + potential gradient as an energy source. This link is tenuous, however, as the overall structure of flagellar motors is far more complex than that of the F O particle and the ring with ca. 30 rotating proteins is far larger than the 10, 11, or 14 helical proteins in the F O complex.!