ATP Production Flashcards
Anaerobic Fermentation
pyruvate + NADH → lactate + NAD+
3 Stages of converting glucose to ATP
Stage 1: The fuel molecules are oxidized to two-carbon fragments in the form of the acetyl group of acetyl~CoA (the ~ bond indicates that this is a high-energy bond, meaning that the hydrolysis of the bond releases a great deal of energy can be used to drive a chemical reaction).
Stage 2: The acetyl groups enter the tricarboxylic acid (TCA) cycle which produces CO2 and the reduced energy carriers NADH and FADH2
Stage 3: Stage 3: The NADH and FADH2 are oxidized in the electron transport chain (ETC), producing H+ and electrons, and then the electrons are transferred to O2,
producing water. The protons are used in a process known as oxidative phosphorylation (OxPhos) to produce ATP
Coenzyme A (CoA)
Coenzyme A (CoA) is a coenzyme that is derived from ATP and the B vitamin pantothenic acid (B5). It contains a reactive –SH (thiol) group that is covalently linked to the acetyl group of acetyl~CoA via a thioester bond. Thioester bonds are relatively high-energy bonds, and so acetyl~CoA can readily donate acetyl groups to other acceptors.
Production of Acetyl~CoA from Pyruvate
Pyruvate is formed in the cytoplasm by glycolysis, and then is transported into the mitochondrial matrix, where it is converted to acetyl~CoA by the pyruvate dehydrogenase (PDH) complex with the release of CO2.
3 types of α-ketoglutarates
Pyruvate Dehydrogenase
Branched chain α-ketoacid dehydrogenase
α-ketoglutarate dehydrogenase
The PDH complex contains three enzymes present in multiple copies:
pyruvate dehydrogenase (aka pyruvate decarboxylase) (E1) dihydrolipoyl transacetylase (E2) dihydrolipoyl dehydrogenase (E3)
Which subunits are identical between members of the α-ketoacid dehydrogenase family?
The E3 subunits are identical between members of the α-ketoacid dehydrogenase family.
What coenzymes/cosubstrates/prosthetic groups are associated with the PDH Complex?
In addition to the E1, E2, and E3 subunits, the PDH complex contains two stoichiometric coenzymes/cosubstrates (NAD, CoA) and three catalytic coenzyme prosthetic groups (thiamine pyrophosphate (TPP), FAD, and lipoic acid).
Overall reaction scheme of oxidative decarboxylation of pyruvate by the PDH complex
- Pyruvate is decarboxylated to form a hydroxyethyl derivative bound to the reactive carbon of thiamine pyrophosphate, the coenzyme of pyruvate decarboxylase (E1)
- The hydroxyethyl intermediate is oxidized by transfer to the disulfide form of lipoic acid covalently bound to dihydrolipoyl transacetylase (E2)
- The acetyl group, bound as a thioester to the side chain of lipoic acid, is transferred to CoA
- The sulfhydryl form of lipoic acid is oxidized by FAD-dependent dihydrolipoyl dehydrogenase (E3) leading to the regeneration of oxidized lipoic acid.
- FADH2 on E2 is reoxidized to FAD as NAD+ is reduced to NADH + H+
Function of long lysine side chain of dihydrolipoyl transacetylase
The long lysine side chain of dihydrolipoyl transacetylase (E2) serves as a swinging arm to transfer electrons and the acetyl group from pyruvate dehydrogenase (E1) to dihydrolipoyl dehydrogenase (E3):
The swinging arm of E2 keeps the acetyl group and the electrons from pyruvate formed by E1 in close proximity to E3. This means that none of the intermediates can diffuse away from the complex during the reaction, markedly enhancing the efficiency of the whole process. This containment of the intermediates within the complex is an example of what is known as substrate channeling.
Niacin and thiamine deficiency
Niacin and thiamine deficiency can cause severe central nervous system deficiency. Niacin is one of the precursors to NAD+/NADH, while thiamine is
the precursor to TPP, the coenzyme for E1. A lack of either NAD+/NADH or thiamine will reduce/block the activity of of the PDH complex, resulting in the eventual reduction in ATP synthesis. The CNS is heavily dependent on ATP to support its high level of activity, and so a reduction in ATP synthesis means reduced CNS function.
Arsenic
Arsenic in the form of trivalent arsenite forms a stable complex with the thiol groups of lipoic acid, rendering it unable to serve as a coenzyme for PDH.
Regulation of the PDH Complex: activation of E1
E1 is inactivated by phosphorylation by PDH kinase, and E1 is activated by dephosphorylation by PDH phosphatase.
Positive Allosteric Effectors of PDH Kinase
ATP, Acetyl CoA and NADH
Negative Allosteric Effectors of PDH Kinase
Pyruvate
Positive Allosteric Effectors of PDH Phosphatase
Ca2+
Negative Feedback Inhibition of PDH Complex
NADH and Acetyl CoA
Step 1 of TCA Cycle: Formation of citrate
The condensation of acetyl~CoA and oxaloacetate (OAA) is catalyzed by citrate synthase. The reaction is made irreversible by the hydrolysis of the thioester bond if
acetyl~CoA and facilitated by an enzyme-bound intermediate, citroyl~CoA. The CoA liberated in this reaction can then be used by the PDH complex for oxidative decarboxylation of another pyruvate.
Step 2 of TCA Cycle: Isomerization of citrate to isocitrate
Citrate is isomerized to isocitrate by the enzyme aconitase via a cis-aconitate intermediate. The equilibrium of the reaction is actually towards the left (formation of citrate (note the (+)ve ∆Go), but since isocitrate is rapidly consumed in the net step, the reaction is pulled to the right in vivo.
Step 3: Oxidation of isocitrate to α-ketoglutarate and CO2
Isocitrate dehydrogenase catalyzes the irreversible decarboxylation of isocitrate, along with the generation of NADH. Due to the loss of CO2, this is an irreversible reaction
Step 4: Oxidation of α-ketoglutarate to Succinyl~CoA and CO2
This reaction is catalyzed by the α-ketoglutarate dehydrogenase complex, which is very similar to the PDH complex and uses the same coenzymes/cosubstrates (NAD, CoA) and catalytic coenzyme prosthetic groups (thiamine pyrophosphate (TPP), FAD, and lipoic acid) as PDH.
This reaction is irreversible and is another site of generation of NADH. Isocitrate dehydrogenase and α-ketoglutarate dehydrogenase are responsible most of the CO2 produced and exhaled.
Step 5: Conversion of succinyl~CoA to succinate
The thioester bond in succinyl~CoA is a high-energy bond and hydrolysis of this bond can be used to drive other reactions. In this case succinate thiokinase (aka succinylCoA synthetase) couples the cleavage of the thioester bond to the formation of a highenergy phosphate bond in the conversion of GDP to GTP:
The GTP produced is another example of substrate-level phosphorylation, such as was seen in glycolysis. GTP can be used to drive the phosphorylation of ATP by nucleoside diphosphate kinase, so GTP and ATP are energetically interconvertible:
Step 6: Oxidation of succinate
Succinate is oxidized to fumarate by succinate dehydrogenase with the concomitant production of FADH2. Succinate dehydrogenase is unique among the enzymes of the TCA cycle is that it is not a soluble enzyme but rather is localized to the mitochondrial inner membrane.
Steps 7 & 8: Regeneration of oxaloacetate
The last two steps in the cycle involve the conversion of fumarate to malate by fumarase followed by conversion of malate to OAA by malate dehydrogenase with the
generation of the last of the three NADH generated by one turn of the cycle.
Summary of the TCA Cycle
Two carbons enter the TCA cycle in the form of an acetyl group and two leave in the form of 2 CO2 molecules.
The energy released in the reactions is conserved in the reduction of 3 NAD+ and 1 FAD and the production of 1 GTP.
The NADH and FADH2 produced with each turn of the cycle will be used in the electron transport chain to generate ATP via oxidative phosphorylation.
Three irreversible steps (citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) prevent the cycle from reversing direction.
Regulation of citrate synthase
Citrate synthase is inhibited by citrate via competitive inhibition.
Regulation of Isocitrate dehydrogenase
Isocitrate dehydrogenase is inhibited by ATP and NADH, and stimulated by ADP and Ca++
Regulation of α-ketoglutarate dehydrogenase
α-ketoglutarate dehydrogenase is inhibited by succinyl~CoA and NADH and activated by Ca++. Although similar in structure to the PDH complex, it is not regulated by phosphorylation/dephosphorylation.
Complex I: NADH dehydrogenase
This enzyme contains a tightly bound flavin mononucleotide (FMN) coenzyme (similar in structure to FAD) that can accept two electrons from NADH to become FMNH2 and then from FMNH2 to CoQ to form CoQH2. The enzyme also contains iron-sulfur (Fe-S) centers which participate as intermediate electron carriers in this process.
Complex II: Succinate dehydrogenase
This is part of the TCA cycle which oxidizes succinate to fumarate. Electrons from succinate move via FAD and Fe-S centers to CoQ to form CoQH2. CoQ/CoQH2 serves as a mobile electron carrier that can shuttle electrons between different parts of the ETC.
Complex III: Cytochrome bc1
Cytochromes contain a heme group in which the iron shifts from the Fe+3 to Fe+2 oxidation states and back as electrons move to and from the heme.
Like Complex I and Complex II, Complex III also contains Fe-S centers.
In Complex III, electrons from CoQH2 are used to reduce cytochrome c, a soluble protein which is located in the intermembrane space and serves as a mobile electron carrier
Complex IV: Cytochrome oxidase
This contains two different heme groups (hemes a
and a3) and two Cu ions that form a complex similar to the Fe-S centers seen in Complexes I and II. Electrons from cytochrome c are transferred to heme a via one of
the Cu centers, from heme a to heme a3 via the other Cu center, and then to O2 to form H2O.
Rotenone
Inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone, This interferes with NADH during the creation of usable cellular energy (ATP)
Antimycin A
Inhibits the reduction of ubiquinone in the electron transport chain of oxidative phosphorylation.
CN- or CO
Binds to cytochrome c oxidase in the electron transport chain. It attaches to the iron within this protein complex and inhibits the normal activity of the complex system. It binds tightly so that it cannot transport any electrons to oxygen, thereby unable to become oxidized.
For each pair of electrons that travel through the ETC and are transferred to O2, how many protons are pumped?
For each pair of electrons that travel through the ETC and are transferred to O2, 4 protons are pumped out by Complex I, 4 by complex III, and 2 by Complex IV, for a total of 10 protons.
How do mitochondria deal with reactive oxygen stress?
Mitochondria have evolved systems to eliminate these free radicals that use the enzyme superoxide dismutase to convert • O2 to peroxide (H2O2), and then glutathione peroxidase to convert peroxide to water.
P:O ratio
The amount of ATP formed per ½ O2 consumed (or
per pair of electrons). The P:O ratio approaches 3 for NADH oxidation, while that for succinate oxidation is closer to 2, since succinate oxidation goes via Complex II and bypasses Complex I.
How are ATP synthesis & electron transport coupled?
Addition of ADP induces a large increase in O2
consumption as electrons flow though the ETC; in addition, an increase in ATP would also be detected. When ADP is depleted, the rate of O2 consumption decreases; addition of additional ADP accelerates the process again. The coupling of electron transport and ATP synthesis is termed respiratory control.
Chemiosmotic Hypothesis
The proton motive force drives ATP synthesis as the protons flow passively back through the inner mitochondrial membrane down their concentration gradient through a proton pore in the ATP synthase
Mitochondrial ATP synthase
The mitochondrial ATP synthase (aka Complex V or F1Fo ATPase). The F1 portion contains the ATPase domain, and the Fo portion contains the transmembrane proton pore.
Mitochondrial ATP synthase: F1 portion
F1 contains 9 subunits in the stoichiometry α3β3γδε, where the α and β subunits form the knoblike catalytic section and γ subunit forms a shaft that connects with Fo.
Mitochondrial ATP synthase: F0 portion
The Fo complex consists of a, b, and c subunits. The c subunits form a ring called the c-ring; there are 8-15 c subunits on the c-ring depending on the species; in yeast there are 10, while in vertebrates there are 8 subunits per ring. Rotation of the c-ring is induced by protons moving through the pore, and this in turn induces conformational changes in the β subunits of F1 that drive ATP synthesis. One full rotation of the c-ring with x subunits will move x protons across the membrane and produce 3 ATPs.
Yield of ATP Production
From 1 molecule of glucose:
Glycolysis: generates 2 net ATPs + 2 NADH
PDH: generates 1 NADH/pyruvate → 2 NADH/glucose
TCA: generates 3 NADH/pyruvate → 6 NADH/glucose
1 FADH2/pyruvate → 2 FADH2/glucose
1 ATP/pyruvate → 2 ATP/glucose
Oxidative Phosphorylation converts 10 NADH → 27.5 ATP and 2 FADH2 → 3.3 ATP
~31 ATP (OxPhos)
+ 2 ATP (glycolysis)
+ 2 ATP (TCA )
Uncouplers
Uncouplers allow protons to flow down their concentration gradient dissipating the
proton motive force. They prevent ATP synthesis, but remove the coupling brake on the ETC so electron transport and O2 consumption proceed unregulated. Examples of uncouplers are the lipophilic weak acid dinitrophenol (DNP) and uncoupling protein 1 (UCP1 aka thermogenin) found in brown fat mitochondria. In both cases the collapse of the proton motive force releases energy in the form of heat rather than ATP.
Adenine Nucleotide
The adenine nucleotide translocase is an antiporter that binds ADP3- in the intermembrane space and exchanges it for ATP4- from the matrix. Since 4 charges move out of the matrix and 3 enter, transport is stimulated by the matrix-negative transmembrane potential.
Atractyloside
a toxic compound found in thistle, inhibits the adenine nucleotide translocase, preventing the synthesis of ATP.
Phosphate Transport
The phosphate translocase is a symporter that moves one H2PO4- and one H+ into the matrix. This transporter uses the proton gradient to drive the transport.
Nicotinamide nucleotide transhydrogenase
The NADPH formed in this reaction can be used by glutathione reductase as part of the previously-described process of removing ROS generated during electron transport. Since NADH is used by electron transport, its level should be related to the potential for ROS generation, and thus production of NADPH by transhydrogenase will increase as more electrons can travel down the ETC.
Respiratory Inhibitors (ex. CN, CO)
Reduce respiration, oxidative phosphorylation and ancillary reactions.
Phosphorylation inhibitors (ex. oligomycin)
Reduce respiration (gradient-making supressed), oxidative phosphorylation but leave ancillary reactions alone.
Uncouplers (DCP, UCP)
Reduce oxidative phosphorylation and ancillary reactions but leave respiration alone.
Malate-aspartate shuttle
The reducing equivalents of
NADH are transferred to cytosolic oxaloacetate using cytosolic malate dehydrogenase to form malate. The malate then enters the matrix via the malate-α -ketoglutarate. transporter. Once inside the matrix, the malate is converted back to oxaloacetate by
mitochondrial malate dehydrogenase with the concomitant reduction of NAD+ to NADH.
The matrix oxaloacetate is then converted to aspartate and sent back out to the cytosol, where it can be metabolized to oxaloacetate, completing the cycle.
Glycerol-3-phosphate shuttle
In skeletal muscle and brain, the glycerol-3-phosphate shuttle is used. In this shuttle the NADH generated in glycolysis is used to produce glycerol-3-phosphate from
dihydroxyacetone phosphate by the cytosolic enzyme glycerol-3-phosphate dehydrogenase. Glycerol-3-phosphate is converted back to DHAP by the mitochondrial glycerol-3-phosphate dehydrogenase, producing FADH2, which then enters the ETC at
CoQ. Note that since FADH2 is produced instead of NADH, the yield of ATP via this shuttle is less than from the malate-aspartate shuttle.
