Metabolism is regulated by the rate of ATP production

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BCHM2972 Human Biochemistry Introduction to Metabolism Metabolism is regulated by the rate of ATP production Anabolism/Catabolism Anabolism Reactions that build macromolecules Use energy from

1 BCHM2972 Human Biochemistry Introduction to Metabolism Metabolism is regulated by the rate of ATP production Anabolism/Catabolism Anabolism Reactions that build macromolecules Use energy from catabolism Require energy Catabolism Reactions that break things down Liberate energy when monomers are broken down into simple organics ATP Facts Hydrolysis of two terminal phosphates releases a lot of energy o ATP ADP and ADP AMP Energy released as heat unless captured to do something useful o Anabolic reactions! Cell [ATP] = ~5mM o Stored as fuels: carbs, fats, proteins (hydrolysis of protein is last resort) o Cannot synthesize ATP to store for later use Cellular [ATP] must not go below 3mM o Cells die because insufficient ATP for essential housekeeping roles Muscle Cells have ATP stores of about 8mM 8mM = 8umol/g tissue Critical level of ATP is always about half of resting level o Critical level = min. conc. Before death ATP > ADP > AMP Cells respond to [AMP] and [ADP] more exquisitely than [ATP] o ATP levels have to stay constant and would be a poor indicator cell happiness o Important for communicating energy charge information of cell ATP is made on site in cell when it is required ATP not transported between cells High turnover rate of ATP o Total amt of ATP in whole body is about 50g VS Total production and destruction of ATP approaches 1kg/kg body weight/day o 1000:1 ATP generation rate must absolutely match ATP consumption rate

ATP is stable ATP:ADP Ratio is kept high = higher free energy release = [ADP] controls rate of metabolism Hydrolysis of ATP is under enzyme control ATP is the intermediary between anabolism and

2 ATP is stable ATP:ADP Ratio is kept high = higher free energy release = [ADP] controls rate of metabolism Hydrolysis of ATP is under enzyme control ATP is the intermediary between anabolism and catabolism o It is not the most energy- containing molecule in metabolism In a reaction that uses ATP, 40% is lost as heat and 60% is used for useful work Overview of Fuel Oxidation If ATP is needed, it must be made Rate of fuel oxidation (catabolic) is linked to the rate of ATP production o Carbon atoms become CO 2 and Hydrogen atoms become H 2 O Fatty acids and Carbohydrates are very reduced o CO 2 very oxidized Fuel Oxidation Fuel is oxidized o H and e - are ripped out o Turns C in carbs and fat to CO 2 Oxidizing Agent is also an electron carrier protein Nicotinamide Adenine Dinucleotide (NAD) o Flavin Adenine Dinucleotide (FAD) o Rips out H and e - from fuels and carry them around o NAD + (oxidized) + 2H + + 2e - NADH (reduced) + H + {Reduction o 1H + and 2e - are carried on its ring o Niacin (a vitamin) aids in keeping up supply of nicotinamide to have enough NAD Primary Oxidative Pathways Strip out Hs o Carbs Glycolysis o Fatty Acids β- oxidation Sulfur group is responsible for linking CoA to 2- carbon acetate units from oxidation o CoA has a adenine nucleotide and ribose 3 phosphate

All oxidation pathways ultimately end up with acetate groups (Acetyl- CoA), which are funnelled into a pathway Acetyl- CoAs enter the Krebs cycle

(and other carriers) are reacted with Oxygen WATER o Controlled, stepwise manner in which energy is harnessed as ATP NADH passes e - to chain of

from matrix to cytoplasm Proton gradient is generated: Charge & Chemical NAD is regenerated Proteins in ETC Complexes 1 4 o 2 Classes of Carriers

Structural: Maintain shape of complex o Prosthetic Group: Transporter of H/e - Proteins are arranged such that o H + expelling reactions are on

3 All oxidation pathways ultimately end up with acetate groups (Acetyl- CoA), which are funnelled into a pathway Acetyl- CoAs enter the Krebs cycle Pathway strips out H via NAD Fully oxidizes the carbons in acetyl- CoA NADH and CO 2 are generated Electron Transport Chain Hydrogens carried on NADH (and other carriers) are reacted with Oxygen WATER o Controlled, stepwise manner in which energy is harnessed as ATP NADH passes e - to chain of transporters in inner mitochondrial membrane o Impermeable to protons duh Final electron carrier: Oxygen As Hs move down chain, protons are pumped from matrix to cytoplasm Proton gradient is generated: Charge & Chemical NAD is regenerated Proteins in ETC Complexes 1 4 o 2 Classes of Carriers Carries exclusively e - Carries mixture of e - and H + (H or H - ) o UQ = Ubiquinone o Cytochrome C Each complex consist of many proteins o Structural: Maintain shape of complex o Prosthetic Group: Transporter of H/e - Proteins are arranged such that o H + expelling reactions are on outside (IMS side) o H + consuming reactions are on matrix side 1 NADH = about 10 H + Note: Not all the protons originate from the particular NADH, but ultimately from everywhere

NADH: o I III IV o Carries H + with 2e - = H - (hydride ion) o Thermodynamically same with NADP+ Recognized by diff enzymes FADH 2 : II III IV o Acceptor of only H FAD + +2 H + + 2e - FADH2 o

4 NADH: o I III IV o Carries H + with 2e - = H - (hydride ion) o Thermodynamically same with NADP+ Recognized by diff enzymes FADH 2 : II III IV o Acceptor of only H FAD + +2 H + + 2e - FADH2 o Completely immobilized in Complex II Not free in cytosol like NAD+ Complexes I, Ubiquinone (Coenzyme Q) Immobilized in membrane: very hydrophobic! Accepts Hs from Complex II (H + + e - ) o Reduction from UQ to UQH 2 UGH 2 transfers Hs to Complex III from II or I Cytochrome C & Fe- S: ONLY DEAL WITH e - Accepts e - from Complex III and transfers to Complex IV Prosthetic group that contains Fe o Fe 3+ to Fe 2+ and back o Carriers that usually have Fe or Fe- S Complex porphyrin ring structure

5 Actual Pumping Alternating complexes that deal with H only or electrons only e - are always flowing down the chain uninterrupted When a complex deals with only H, a H + is sucked in from the matrix When the next complex only deals with e -, a H + is released into the IMS The orientation of the uptake/release can allow net translocation (pumping o Proton releasing reactions on the cytoplasmic side o Proton consuming reactions on the matrix side Inhibitors and Acceptors Rotenone o Inhibits Complex I o Entire ETC stops, H+ pumping stops o All complexes downstream are oxidized Cyanide o Inhibits Complex IV o Entire ETC stops, H+ pumping stops o All complexes upstream remains reduced Does not matter which complex is blocked, the whole ETC halts since flow of electrons stop o All complexes downstream are oxidized (electrons are lost) o All complexes upstream reduced Alternative Acceptor E.g. Methylene Blue o Accepts electrons from Complex IV o Allow electron transport to continue if there is steady supply of the compound o Continued flow of electrons from I to IV and pumping still occurs Oxidative Phosphorylation: ATP Generation ATP Synthase: F 0 F 1 ATPase F 0 : 12 cylindrical proteins make up channel Pore in inner mitochondrial membrane Protons flow through channel and Rotates ϒ- subunit

F 1 : Each groove rotation of ϒ- subunit changes the conformation of each α & β subunit relative to each other Sequential operation of each α & β subunit

Stator b 2 of F 1 ATPase subunit holds the α & β subunit complexes in place Stoichiometry: How many ATP per proton?

diffuse out of IMS Uncoupling proteins present in all cells can allow protons to move across inner membrane into matrix Protons are used for other

6 F 1 : Each groove rotation of ϒ- subunit changes the conformation of each α & β subunit relative to each other Sequential operation of each α & β subunit Open: ADP + Pi enter α & β subunit pair. There are α & β subunit Squeeze: ADP + Pi squeezed together α & β subunit Open: ATPi released 6 in F 1. Stator b 2 of F 1 ATPase subunit holds the α & β subunit complexes in place Stoichiometry: How many ATP per proton? Movement of 3 protons generates 1 ATP o 1 NADH 10 protons pumped to IMS ~3ATP Things to consider Outer mitochondrial membrane is leaky: protons can diffuse out of IMS Uncoupling proteins present in all cells can allow protons to move across inner membrane into matrix Protons are used for other processes o E.g. Transporting P into IMS uses protons Proton gradient strength can decrease during ATP synthesis o ADP 3- ATP 4- and is transported into cytosol by antiporter o Lessens the effect of the proton gradient

Therefore it takes a little more than 3 protons to generate 1 ATP Consider cytosolic NADH (big, charged, cannot cross membrane on its own) Shuttled into matrix 2 key shuttle systems but different

7 Therefore it takes a little more than 3 protons to generate 1 ATP Consider cytosolic NADH (big, charged, cannot cross membrane on its own) Shuttled into matrix 2 key shuttle systems but different efficiency Malate- Aspartate Shuttle 1 for 1 exchange o 1 cytosolic NADH becomes 1 matrix NADH Complete efficiency NADH interacts with Complex 1 10 H+ pumped to IMS from 1 NADH Glycerol 3- Phosphate Shunt Inefficient transport of cytosolic NADH Reacts with protein on surface of membrane (facing IMS) Protein acts similarly to Complex II (contains FAD) Complex 1 is skipped 6 H+ pumped to IMS from 1 NADH Therefore, never can completely accurately calculate # of ATP synthesized per process e.g. glycolysis Note: FA ox used FAD to rip out H and make double bond in FA for glycolysis This will also bypass Complex 1 Overview:

8 Coupling Regulation of Oxidative Phosphorylation I: Movement of protons and synthesis of ATP via ATP synthase cannot occur independently ATP is made when H + comes through the ATP synthase BUT H + will ONLY come in through ATP synthase if ADP and Pi are available and are being made into ATP II: Movement of H/e - down ETC and H + movement from matrix IMA cannot occur independently Proton gradient is made as H/e - move down the ETC o 1H from NADH 10H+ Electrons can only flow down ETC ONLY if protons are simultaneously pumped out III: The bigger the proton gradient = harder to pump protons out of matrix to IMS Bigger the proton gradient = slower movement of H/e + down ETC from NADH to oxygen IV: Carriers need to be regenerated NAD: Carrier of 1H + and 2e - ADP: Carrier of phosphate Complexes I to IV, UBQ, Cyt C Carriers need to release their cargo (oxidized) to become available again

9 Scenarios No Work Extreme and never really happens No consumption of ATP = No regeneration of ADP o Remember that ATP is stable and will only break down if told by enzyme No ADP = No substrate for ATP synthesis No ATP synthesis = Proton gradient is not dissipated and cannot pass through ATP synthase High H + = No pumping of H + across ETC No pumping across ETC = No H/e - flow down ETC = ETC STOP No ETC flow = NADH cannot oxidize and pass H/e - = No NAD regenerated No NAD = No fuel oxidation No fuel oxidation = No oxygen consumption Doing Work ATP consumed = Regeneration of ADP H + flow through ATP Synthase Proton gradient pressure relieved Proton pumping resumes H/e - move down ETC to oxygen NADH can oxidize to NAD NAD available for fuel oxidation Rate of fuel oxidation matches rate of ATP consumption o Rate of oxygen consumption good indicator of fuel ox rate and energy expenditure Uncoupling: Dissipating proton gradient w/o making ATP If protons do not return to matrix via ATP synthase Proton gradient o Protein carrier No proton gradient dissipates = No driving force for ATP synthase = No ATP synthesized SHORT CIRCUIT Proton pumping across ETC continues NADH continues to oxidize and regenerate NAD Massive fuel ox. rate and oxygen consumption ATP levels suffer Cell death Energy dissipated as HEAT Dinitrophenol Hydrophobic and can move freely across membrane Weak acid o In acidic environment: Bind to proton o In alkaline environment: Releases proton Dissipates proton gradient by shuttling protons from IMS back to matrix

Oxidative Phosphorylation

Oxidative Phosphorylation Electron Transport Chain (overview) The NADH and FADH 2, formed during glycolysis, β- oxidation and the TCA cycle, give up their electrons to reduce molecular O 2 to H 2 O. Electron transfer occurs through