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Pyruvate dehydrogenase complex
A complex that converts pyruvate to acetyl CoA; it produces CO2 and captures high-transfer-potential electrons in the form of NADH; large, highly integrated complex of three distinct enzymes; member of a family of homologous complexes that include the CAC enzyme alpha-ketoglutarate dehydrogenase complex; contains pyruvate dehydrogenase complex, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase
Pyruvate dehydrogenase component
24 chains; TPP; catalyzes the oxidative decarboxylation of pyruvate
24 chains; lipoamide; transfers the acetyl group to CoA
12 chains; FAD; regenerates the oxidized form of lipoamide
Enzyme that catalyzes the condensation of oxaloacetate with acetyl CoA to form citrate; dimer with active site located in cleft between large and small domains of subunit; undergoes large conformational changes in catalysis; exhibits ordered, sequential kinetics: oxaloacetate first binds, then acetyl CoA. The binding of oxaloacetate converts the open form into the closed form. The small domain rotates; and movements are produced by rotation of the alpha helices; these changes create a binding site for acetyl CoA
Catalyzes isomerization of citrate into isocitrate to allow it to undergo oxidative ecarboxylation; accomplished by a dehydration and then hydration; iron-sulfur protein, containing Fe-S clusters that participate in dehydrating and rehydrating the bound substrate
Alpha-ketoglutarate dehydrogenase complex
Enzyme that complexes the decarboxylation of alpha-ketoglutarate to succinyl CoA
Succinyl CoA synthetase
Catalyzes the coversion of succinyl CoA into succinate
Catalyzes the transfer of a phosphoryl group from GTP to ADP, forming GDP and ATP
Iron-sulfur protein with three different kinds of iron-sufur clusters; embedded in inner mitochondrial membrane and directly associated with the ETC, the link between the CAC and ATP formation. FADH2 produced by oxidation of succinate does not dissociate from the enzyme, in contrast with NADH produced in other oxidation-reduction reactions. The two electrons are transferd from FADH2 directly to Fe-S clusters and then to CoQ
A neurologic and cardiovascular disorder caused by dietary deficiency of thiamine
1) What is the function of the CAC in transforming fuel molecules into ATP?
- a. Fuel molecules are carbon compounds that are capable of being oxidized—that is, of losing elecctrons. The CAC includes a series of redox reactions that result in the oxidation of an acetyl group to two molecules of CO2. This oxidation generates high-energy electrons that will be used to power the synthesis of ATP. So, the CAC harvests high energy electrons from carbon fuels.
- b. It removes electrons from acetyl CoA and uses these electrons to form NADH and FADH2
1) What are the steps of the pyruvate dehydrogenase complex?
- a. Decarboxylation: pyruvate combines with TPP and is then decarboxylated to yield hydroxyethy-TPP
- b. Oxidation: the hydroxyethyl group to TPP is oxided to form an acetyl group and is transferred to lipoamide, a derivative of lipoic acid that is linked to the side chain of a lysine resiude by an amide linkage. This leads to the formation of an energy-rich thioester bond.
- c. Formation of the acetyl CoA: the acetyl group is transferred from acetyllipoamide to CoA to form acetyl CoA; E2 catalyzes this reaction; and, the thioester bond is preserved.
- d. The oxidized form of lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3). Two electrons are transferred to an FAD prosthetic group of the enzyme and then to NAD+, an unusual exchange because FAD usually accepts proteins
1) What is the structure of the complex?
- a. The core is formed by the transacetylase component E2, which has 8 catalytic trimers assembled to form a hollow cube; each subunit forms a trimer with three major domains. At the N-terminus is a small domain wth a flexible lipoamide domain followed by a small domain that interacts with the E3 complex. A larger transacetylase domain completes an E2 subunit.
- b. E1 is an alpha2beta2 tetramer; E3 is an alphabeta dimer
1) How does citrate synthase catalyze the condensation reaction?
a. It brings the subunits into close proximity, orienting them, and polarizing certain bonds. Acetyl CoA is transformed into an enol intermediate, which attacks oxaloacetate to form a C=C linking acetyl CoA and oxaloacetate. The active site then becomes completely enclosed. The citryl CoA thioester is then cleaved
1) Explain what the cleavage of the thioester bond of succinyl CoA does?
a. It is coupled to the phosphorylation of a purine nucleoside diphosphate, usually ADP. This reaction is catalyzed by succinyl CoA synthetase.
1) Explain the structure of succinyl CoA synthetase.
a. It is an alpha2beta2 heterodimer. Each subunit has two domains. The amino-terminal domains have different structures. The amino-terminal domain of the alpha subunit forms a ROssman fold, which binds the ADP substrate of succinyl CoA synthetase. The amino terminal domain of the beta subunit is an ATP-grasp domain, which binds and activates ADP
1) What are the final steps after succinate is formed?
- a. Regeneration of oxaloacetate
- i. Succinate is oxidized to fumarate by succinate dehydrogenase, which passes electrons to FAD.
- ii. Then, fumarate is hydrated to form L-malate by fumarase. Finally, malate is oxidized to form oxaloacetate by malate dehydrogenase, and NAD+ is the hydrogen acceptor.
1) What is the end result of the CAC?
- a. Two carbon atoms enter and two carbons leave as CO2.
- b. Four pairs of H atoms leave n four oxidation reactions.
- c. Two NAD+ molecules are reduced in the oxidative decarboxylations of isocitrate and alpha-etoglutarate, one FAD molecule is reduced in the oxidation of succinate, and one NAD+ is reduced in the oxidation of malate.
- d. One compound of ATP is generated from cleavage of succinyl CoA
- e. Two waers are consumed.
1) Explain how they determined whether or not the two atoms were the same.
a. Through isotope-labeling studies, they were were able to show that the two carbon atoms that enter each cycle are not the ones that leave in one cycle despite the symmetry of the molecule.
1) How is oxaloacetate replenished?
a. Oxaloacetate is formed by the carboxylation of pyruvate, in a reaction catalyzed by puyruvate carboxylase. It is active only in the presence of acetyl CoA, which signifies the need for more oxaloacetate. If the energy is high, it is coverted into glucose. If the energy charge is low, it replenishes the CAC
1) What biochemical processes must be affected by deficiency of thiamine?
a. TPP is a prosthetic group of pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and transketolase. The common feature of enzymatic reactions utilizing TPP is the transfer of an activated aldehyde unit. In beriberi, the levels of pyruvate and alpha-ketoglutarate in the blood are higher than normal.
1) What happens to people exposed to mercury or arsenite?
a. The binding of mercury or arsentite to the dihydrolipoyl groups inhibits the complex and leads to the CNS pathologies. Treatment of these poisons is the administration of sulfhydryl reagents with adjacent sulfhydryl groups to compete with the dihydrolipoyl residues for binding with the metal ion. The reagent-metal complex is then excreted in the urine.
1) What are the points of control in the CAC?
a. One is the pyruvate dehydrogenase complex before the CAC. There is also control at isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase.
1) Explain control at the pyruvate dehydrogenase complex.
- a. Because the formation of acetyl CoA from pyruvate is irreversible, there is control here.
- b. High concentrations of reaction products inhibit the reaction: acetyl CoA inhibits the transacetylase component (E2) by binding directly, whereas NADH inhibits the dihydrolipoyl dehydrogenase (E3). This signals that there is no need to metabolize pyruvate to acetyl CoA because the energy needs are being met. This spares glucose.
- c. Covalent modification of E1 by pyruvate dehydrogenase kinase I (PDK) switches off the activity of the complex. Deactivation is reversed by the pyruvate dehydrogenase phosphoatase. It is switched off when the energy charge is high.
- i. ADP and pyruvate activate the dehydrogenase by inhibiting the kinase. The phosphatase is stimulated by Ca2+, the same signal that initiates muscle contraction.
1) How is the CAC controlled?
- a. The first control site is isocitrate dehydrogenase, which is allosterically stimulated by ADP, enhancing its affinity for substrates. The binding of isocitrate, NAD+, Mg2+, and ADP is cooperative. ATP is inhibitory, as is NADH.
- i. This leads to a buildup of citrate, which is transported to the cytoplasm, where it signals phosphofructokinase to halt glycolysis and where it can serve as a source of acetyl CoA for fatty acid synthesis.
- b. The second control site is alpha-ketoglutarate dehydrogenase. It is inhibited by succinyl CoA and NADH, the products of the reaction it catalyzes. The alpha-ketoglutarate that accumulates can beused as a precursor for several amino acids and purine bases.