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Biochemistry 2280A
Chris Brandl

Topic 15 – ATP Synthesis - Proton concentration is about 10 folds higher in intermembrane than matrix (Higher pH matrix) - Excess positive charge builds up in intermembrane - Protonmotive force: combination of proton concentration and electrical potential cause protons to flow back into matrix if given opportunity. This force used to generate ATP  Major driving force is membrane potential Chemiosmotic Hypothesis: protonmotive force drives synthesis of ATP. Evidence: intact membranes required for ATP synthesis ad if make membrane permeable to protons, ATP synthesis stops because destruction of proton gradient. Creating artificial proton gradient stimulates ATP synthesis F1 0ATP Synthase - protein complex that allows protons to flow down electrochemical gradient - energy from proton movement coupled to make ATP in matrix  there is a spatial separation from where protons move to where ATP synthesis occurs - Rotational mechanisms  protons moving cause central piece to turn, which eventually makes ATP - coupling factor 1 “F1” is attached to transmembrane segment “F0” that allows protons to move across membrane - human enzyme is very complex with many distinct subunits Mechanism of E coli ATP Synthase - F0 3 subunits ab c 2 10proton channel (allows p+ to move across membrane) - F1 5 subunits α β3 3ε and makes ATP  Membrane peripheral proteins bound to F0  Proton binds to particular residue of c partway across membrane - Each c subunit binds one proton from intermembrane space - Ring of c rotates, moving p+ to pathway formed with a subunit, allowing protons to be released into matrix - As c rotates, γ and ε subunits (central stalk) rotate along it - Two long, curved α-helices from γ subunit extend into α β 3 3 hexamer 1 - As γ rotates, causes changes in conformation of β subunits – each contain catalytic site for ATP synthesis. Conformational changes drive synthesis of ATP  γ in middle of αand β s, as γ rotates, bumps against β in different way because γ is bent. As a result, β changes shape  ATP is synthesized - α3 3hexamer is prevented from rotating by “peripheral stalk” composed of 2 b subunits and δ - b subunits anchored to a subunit in membrane and δ helps bind b to α β 3 3  ATP is rotary motor, with rotor composed of c γε10nd stator composed of abα β3 3 Role of Each subunit F 0 F1  α β – form active sites (one on each 3 3 β); conformational changes cause ATP  a – helps form proton channel; anchors synthesis b in membrane  γ – forms part of central stalk; rotates 2  b –2forms second stalk (the “stator”) with c 10 cause conformational that prevents α β3 3om rotating changes in α 3 3  δ – helps attach b to α β  c –10elps form proton channel; 2 3 3 rotates during proton movement  ε – helps assemble complex; inhibits ATP hydrolysis (reverse process) Net Reaction - For each full rotation of c γε ring 10  10 protons cross membrane  3 ATPS made (because γ contacts 3 different β sites) - Synthesis of 1 ATP requires 3⅓ protons to move across membrane - number of c subunits in ATP synthase vary between organisms, so number protons moved per ATP synthesized may also vary 3 ADP + 3Pi + 10H+intermembrane  3ATP + 3H2O + 10H+matrix Import and Export - ATP needs to be moved from matrix into cytosol to be useful and ADP and Pi need to move into matrix for ATP synthesis  One transporter exchanges ATP from matrix for ADP from cytosol  ATP more negative so favoured by electrical potential across membrane 2  Second transporter transports H2PO4- with proton into matrix  uses proton concentration gradient Stoichiometry of ATP Synthesis - Assume synthesis of 1 ATP uses 3 H+ across membrane  Synthesis and export of ATP requires 4 H+ (additional 1 to move ATP, ADP and Pi across membrane) - Oxidation matrix NADH pumps 10 protons  makes 2.5 ATP - Oxidation FADH2 or cystolic NADH pumps 6 protons  makes 1.5 ATP Complete ATP yield from oxidation of Glucose  Glycolysis: 2 ATP, 2 NADH (cystolic)  2 + 3 = 5 ATP  Pyruvate oxidation to acetyl-coA: 2 NADH  5 ATP  Citric Acid Cycle: 2 ATP, 6 NADH, 2 FADH2 2 + 15 + 3 = 20 ATP TOTAL: 30 ATP SUMMARY + •Electrical potential and [H ] gradient across membrane are used to make ATP •F1 0ATP synthase allows protons to enter the matrix, driving ATP synthesis •Aerobic respiration produces ~30 ATP per glucose molecule, versus 2 for anaerobic glycolysis Topic 16: Acetyl-CoA part 2: Fatty acids, Ketone bodies, and biosynthesis - Energy needed from fat storage, lipase in adipocytes degrade triacylglycerol into glycerol and 3 fatty acids - Fatty acids enter blood stream and used as fuel in cells such as skeletal muscle, heart, and liver (not brain) and converted to acetyl-coA via B-oxidation 3 - Glycerol substrate goes to liver for gluconeogenesis - B-Oxidation of Fatty acids - Using fatty acids for fuel, specific transporters move fatty acid from blood into cytosol in cell  Coenzyme A is a carrier that functions to move fatty acid into the matrix - Fatty acid activated with CoA in cytosol in a reaction driven by hydrolysis of ATP to AMP and PPi - Resulting acetyl-CoA oxidized in series of 4 chemical reactions that each remove 2C and produce 1NADH, 1FADH2, and fatty acyl-coA that is 2 C shorter than starting material  Add double bond  reduction makes FADH2  Oxidize alcohol to ketone  reduction makes NADH  Acetyl-coA removes 2 C - Series repeats shortening C by 2 until fatty acid is entirely oxidized to acetyl-coA  For acyl-coA with 2N carbons Acyl-coA + N-1 FAD + N-1 NAD + N-1CoA  N Acetyl-coA + N-1 FADH + N-1 NAD2 - reactions occur in matrix so acetyl-coA and NADH can be used directly by citric acid cycle and FADH2 and NADH donate electrons to ETC  FADH2 gives electrons to Q to make QH2, but using a different protein other than complex II β-oxidation of fatty acids: special cases 4  can accommodate unsaturated fatty acids, but yields fewer FADH2 and may consume NADPH o this is because one step is to make double bond, so if double bond already in right spot, can skip that step  don’t make FADH2  fatty acids with odd number C produce 1 propionyl-CoA in final step  converted to succinyl-coA and can enter citric acid cycle Application – Newborn screening - some people cant oxidize or uptake fatty acids  fatty acids build up in blood stream  leads to seizures, coma, or death - In Ontario, screen babies for fatty acids in blood stream, if high concentration detected, parents notified because condition can be controlled with proper diet Ketone Bodies - Liver mitochondria can convert acetyl-coA to ketone bodies which are then exported from liver and travel through blood to other tissues (brain, muscle) where cells convert back to acetyl-coA to make ATP - Occurs during low glucose conditions  Low glucose: liver breaks down glycogen to glucose  Liver does gluconeogenesis breaking down protein  can’t do that forever because need protein  Use ketone bodies for energy instead Fatty Acid Biosynthesis: - If energy supply is high (high glucose conditions), acetyl CoA from glycolysis is converted to fatty acids in cytosol because cells can only store so much glycogen - Tricarboxylate Transport System  Moves Acetyl-coA from matrix to cytosol  consumes ATP and NADH but produces NADPH - Fatty acid Synthase links 2 C units of acetyl coA in cytosol  Similar to B-oxidation by in reverse  Consumes NADPH and 1 ATP 5  Most commonly produces palmitate (16C:0) – 16 C fatty acid 8 Acetyl-CoA + 14NADPH + 7ATP  Palmitate + 8Co-A + 14NADPH + 7ADP + 7Pi  Fatty acids of other lengths (2N C) can be made: consume 2N-2 NADPH and N-1 ATP  Enzymes can
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