16.2 Reactions of the Citric Acid Cycle The Sequence of Reactions in the Citric Acid Cycle Make Chemical Sense . Acetyl CoA, an intermediate of the breakdown of carbohydrates, fats, and proteins, must be completely oxidized to CO2 in order to extract the maximum amount of potential energy in the fuel. Glycolysis Pyruvate Dehydrogenase Complex FUEL SOURCE converts fuel into pyruvate converts pyruvate into acetyl-CoA Carbohydrates Fats Proteins… Pyruvate Acetyl-CoA etc..until the fuel is completely oxidized to CO2 The Citric Acid Cycle Has Eight Steps Reactants: 1. Acetyl CoA 2. Oxaloacetate Enzyme: Citrate Synthase Product(s): Citrate By-products (that are released): CoA-SH Type of Reaction: Hydrolysis (that means that H2O must be added so that it can break the Citroyl-CoA (intermediate) up. Free CoA and citrate are formed, which are released from the active site. Where do the released products go? The released CoA is recycled back into the oxidative decarboxylation of another pyruvate molecule in the PDH complex. Exergonic (energy is released) because this reaction is the hydrolysis of a high energy thioester (Citroyl-CoA) intermediate. What drives this reaction? The large, negative standard free energy change is the driving force behind this reaction. Oxaloacetate is present at such a low concentration that it cannot be the one driving the reaction to the products side. ❶ Formation of Citrate ❷ Formation of Isocitrate via cis-Acontitate Reactant(s): Citrate Enzyme: Aconitase What does aconitase do? 1. Aconitase catalyzes the reversible transformation of citrate to isocitrate. A reaction intermediate, cis-Aconitase, forms along the way. 2. Has an iron-sulfur center, which serves two purposes: - It helps the substrate bind to the active site - It helps with the addition and removal of H2O. Product(s): (a) Intermediate Product: cis-Aconitate (b) Final Product: Isocitrate Type of Reaction: (a) Citrate to cis-Aconitase is a dehydration reaction (H2O is removed). – NOTE: cis-Aconitate does not dissociate from the active site usually. (b) cis-Aconitase to Isocitrate is a hydration reaction (H2O is added). Endergonic. What drives this reaction? The next reaction in the citric acid cycle (Rxn. # 3) consumes isocitrate rapidly, so the stead-state concentration of isocitrate is low. Therefore, the reaction gets pushed to the right, towards isocitrate formation. NOTE: This occurs despite the fact that the equilibrium mixture of citrate and isocitrate at pH 7.4 and T = 25 °C is only 10% isocitrate. However, the quick consumption of isocitrate in the following reaction, forces the reaction to the right. ❸ Oxidation of Isocitrate to α-Ketoglutarate and Reactant(s): Isocitrate Enzyme: Isocitrate Dehydrogenase There are two forms of isocitrate dehydrogenase: 1. One requiring NAD+ as an electron acceptor. 2. One requiring NADP+ as an e- acceptor. + Product(s): α-Ketoglutarate, CO2, NAD(P)H + H Type of Reaction: Oxidative Decarboxylation CO2 ❹ Oxidation of α-Ketoglutarate to Succinyl-CoA Reactant(s): α-ketoglutarate Enzyme: α-ketoglutarase dehydrogenase complex (very similar reaction mechanism to pyruvate dehydrogenase complex) α-KETOGLUTARATE DEHYDROGENASE COMPLEX VS. PYRUVATE DEHYDROGENASE COMPLEX Similarities Differences Both complexes have 3 (The three enzymes share enzymes subunits. similarities, but are and CO2 ultimately different structures, except E3?). E1 E1 of both complexes are Their amino acid structurally similar. sequences differ. They have different e.g. both have enzyme binding specificities. bound TPP PDC: E1 binds pyruvate KGDC: E1 binds α- Ketoglutarate. E2 Both have covalently bound lipoyl moieties. E3 Identical Structures. Product(s): Succinyl-CoA, CO2 Type of Reaction: Oxidative Decarboxylation NAD+ serves as the electron acceptor CoA is the carrier of the succinyl group What drives this reaction forward? A large and negative free energy change. α-Ketoglutarate Dehydrogenase Complex ❺ Conversion of Succinyl-CoA to Succinate Reactant: Succinyl CoA Succinyl CoA has a thioester bond (with a very large and negative standard free energy of hydrolysis ΔG’° = -36 kJ/mol). Enzyme: Succinyl-CoA Synthetase This reaction is reversible Product(s): Succinate, GTP, CoA-SH. NOTE: The GTP molecule produced can be considered as an ATP molecule because both are energetically equivalent and are interchangeable as long as the required enzyme is present for the conversion. GTP + ADP GDP + ATP ΔG’° = 0 kJ/mol What drives this reaction: The large, negative ΔG’° of hydrolysis for Succinyl CoA drives the reaction forward. The energy that is released when Succinyl-CoA’s thioester bond breaks drives the synthesis of a phosphoanhydride bond in ATP or GTP, with a net ΔG’° of only -2.9 kJ/mol. ❻ Oxidation of Succinate to Fumarate Reactant: Succinate Enzyme: Succinate Dehydrogenase o Malonate, although usually not present in cells, is a strong competitive inhibitor of succinate dehydrogenase. Its addition to the mitochondria blocks the activity of the citric acid cycle. Product(s): Fumarate, FADH2 ❼ Hydration of Fumarate to Malate Reversible Hydration of Fumarate to L-Malate. Catalyst: Fumarase. Very stereospecific. ❽ Oxidation of Malate to Oxaloacetate The equilibrium of this reaction lies far to the left under standard thermodynamic conditions; however, the highly exergonic citrate synthase reaction (the next step/step ❶) keep consuming oxaloactetate, lowering the concentration of the products side of reaction 8. Thus, the reaction is pulled to the right, towards the formation of oxaloacetate, such that a state of equilibrium can be maintained. BOX 16-2 SYNTHASES AND SYNTHETASES; LIGASES AND LYASES; KINASES, PHOSPHATASES, AND PHOSPHORYLASES: YES, THE NAMES ARE CONFUSING! Enzyme Type Function Example Synthases Catalyze condensation reactions in Citrate Synthase which no nucleoside triphosphate (Citric Acid Cycle, Reaction # 1) (ATP, GTP) is required as an energy source. Synthetases Catalyze condensations that use Succinyl-CoA Synthetase ATP or a nucleoside triphosphate (Citric Acid Cycle, Reaction # 6) as a source of energy for the synthetic reaction. Ligases Catalyze condensations in which 2 DNA ligase – closes breaks in DNA ...from the Latin word “ligare” – to atoms are joined using ATP or molecules. tie together another energy source. Synthetases are Ligases. Lyases Catalyze cleavages (or in the The Pyruvate Dehydrogenase reverse direction, additions) in Complex – cleaves CO2 from which electronic rearrangements pyruvate. occurs Kinases Catalyzes a phosphorylation Hexokinase reaction. Transfers a phosphoryl Glucokinase group from a nucleoside triphosphate (e.g. ATP) to an acceptor molecule. Phosphorylases A phosphate attacks a molecule Glycogen Phosphorylase – and covalently bonds at the point phosphorolysis of glycogen, of bond breakage. producing glucose 1-phosphate. Phosphatases Catalysis of dephosphorylation of a - phosphate ester, with water as the attacking species. Dehydrogenase An enzyme that catalyzes the Citric Acid Cycle: removal of hydrogen from a Isocitrate Dehydrogenase (Rxn. 3) substrate and the transfer of the α-Ketoglutarate Dehdyrogenase hydrogen to an acceptor in an Complex (Rxn. 4) oxidation-reduction reaction. Succinate Dehydrogenase (Rxn. 6) Malate Dehydrogenase (Rxn. 8) TYPES OF REACTIONS THAT OCCUR IN THE CITRIC ACID CYCLE Reaction Description Example Condensation A condensation reaction (i.e. dehydration synthesis) is a chemical reaction in which two molecules or moieties (functional groups) combine to form a larger molecule, together with the loss of a small molecule.[1] Possible small molecules lost are water, hydrogen chloride, methanol, or acetic acid. Dehydration A dehydration reaction is a chemical reaction that involves the loss of a water molecule from the reacting molecule. Hydration A hydration reaction is a reaction where a hydrogen and hydroxyl ion is attached to a carbon in a carbon double bond. Decarboxylation Decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2). Oxidation Loss of electrons Always coupled with a reduction reaction – gain of electrons Substrate-Level Substrate-level phosphorylation is a Phosphorylation type of metabolic reaction that results in the formation of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) by the direct transfer and donation of a phosphoryl (PO3) group to adenosine diphosphate (ADP) or guanosine diphosphate (GDP) from a phosphorylated reactive intermediate. .