Citric Acid Cycle & Carbohydrate Metabolism PDF

The citric acid cycle General description The citric acid cycle (CAC) is also known as the Krebs cycle, Szent-Györgyi- Krebs cycle or the TCA cycle (tricarboxylic acid cycle). The cycle consists of a series of 8 chemical reactions to release stored energy through the oxid...

The citric acid cycle General description The citric acid cycle (CAC) is also known as the Krebs cycle, Szent-Györgyi- Krebs cycle or the TCA cycle (tricarboxylic acid cycle). The cycle consists of a series of 8 chemical reactions to release stored energy through the oxidation of acetyl coenzyme A derived from carbohydrates, fats, and proteins. The citric acid cycle is catabolic, because it involves degradation and is a major free energy conservation system in most organisms. However, several biosynthetic pathways utilize citric acid cycle intermediates as starting material (anabolism). The citric acid cycle is therefore amphibolic (both catabolic and anabolic). Its central importance to many biochemical pathways suggests that it is an ancient component of metabolism. The enzymes of citric acid cycle are localized in the matrix of mitochondria (except for the succinate dehydrogenase: inner mitochondrial membrane). 15 The citric acid cycle accounts for the major portion of carbohydrate, fatty acid, and amino acid oxidation, meanwhile it generates precursors for the biosynthesis of fatty acids, steroids, porphyrins, purine and pyrimidine nucleotides, and certain amino acids. 16  Reactions of the Cycle Citrate synthase catalyzes the condensation of acetyl-CoA and oxaloacetate to yield citrate, giving the cycle its name. The strategy of the cycle's next two steps is to rearrange citrate to a more easily oxidized isomer and then oxidize it. Aconitase isomerizes citrate to isocitrate. 17 Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate to produce the citric acid cycle's first CO2 and NADH. The multienzyme complex α-ketoglutarate dehydrogenase oxidatively decarboxylates alpha-ketoglutarate to succinyl-coenzyme A. The reaction involves the reduction of a second NAD+ to NADH and the generation of a second molecule of CO2. 18  GTP is synthesized by the mammalian enzyme; plant and bacterial enzymes utilize ADP + Pi to form ATP. Succinyl-CoA synthetase converts succinyl-coenzyme A to succinate. The free energy of the thioester bond is conserved by the formation of GTP from GDP and Pi. This reaction is a substrate-level phosphorylation. The remaining reactions of the cycle serve to oxidize succinate back to oxaloacetate in preparation for another round of the cycle. Succinate dehydrogenase catalyzes the oxidation of succinate's central single bond to a trans double bond, yielding fumarate with the concomitant reduction of the redox coenzyme FAD to FADH2. This enzyme is embedded in the inner mitochondrial membrane. 19 Fumarase catalyzes the hydration of fumarate's double bond to yield malate. Malate dehydrogenase reforms oxaloacetate by oxidizing malate's secondary alcohol group to the corresponding ketone with concomitant reduction of a third NAD+ to NADH. The net result of the citric acid cycle One turn of the citric acid cycle results in the ultimate generation of 12 ATPs. The regulation of the citric acid cycle is complex; the rate-controlling enzymes: citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase catalyze the irreversible reactions of the cycle. 20  Electron transport and oxidative phosphorylation The catabolism of carbohydrates, lipids and proteins feeds the citric acid cycle. The citric acid cycle is the major source of the reduced universal electron acceptor molecules NADH and FADH2. The final step of catabolism under aerobic condition is oxidative phosphorylation. During oxidative phosphorylation, NADH and FADH2 give the electrons to be converted back to NAD+ and FAD. The electrons move through multiple transport steps before they reduce the final electron acceptor, oxygen, to form water. The flow of electrons through the electron transport chain is an exergonic process. This exergonic process is coupled with ATP synthesis, which is an endergonic process. In eukaryotic organisms, the members of the electron transport chain and the ATP synthase are localized in the 21 inner membrane of mitochondria.  Components of the electron-transport chain -Four respiratory complexes, embedded in the inner mitochondrial membrane. Each of these complexes consists of several protein components that are associated with a variety of redox-active prosthetic groups with successively increasing reduction potential. Three complexes (I, III, IV) also contain polypeptide chains that are transmembrane proteins and pump protons out of the matrix into the intermembrane space. -Between the respiratory complexes, two molecules, coenzyme Q or ubiquinone (UQ) and the relatively small cytochrome c transfer the electrons. -- Electron donors: NADH + H+ and FADH2, passing two electrons to the chain at a time - Final electron acceptor: O2 22  The oxidized and reduced states of all iron-sulfur cluster differ by one formal charge regardless of their number of Fe atoms. (The individual Fe atoms in the clusters can have the oxidation state +2 or +3.) The three types of iron-sulfur clusters that occur as prosthetic groups of iron-sulfur proteins Each group of cytochromes contains a differentially substituted porphyrin ring coordinated with the redox- active iron atom. These heme groups reversibly alternate between their Fe(II) and Fe(III) oxidation states during electron transport. The chemical structures of the heme groups contained in cytochromes a, b, and c 23  The oxidation states of FMN and CoQ Although NADH can only participate in a two-electron transfer, both FMN and CoQ are capable of accepting and donating either one or two electrons because their semiquinone states are stable. In contrast, the cytochromes of Complex III are only capable of one-electron reductions. FMN and CoQ thereby provide an electron conduit between the two-electron donor NADH and the one-electron acceptors, the cytochromes. 24  What determines the sequence of electron transport? The flow of electrons between two molecules depends on their relative affinity for electrons. The reduction potential measures a molecule’s affinity for electron. An electron flows from an electron donor molecule to an acceptor molecule that has higher reduction potential than the donor. The changes in standard reduction potential of an electron pair as it successively traverses Complex I, III, and IV correspond, at each stage, to sufficient free energy to power the synthesis of an ATP molecule. Standard reduction potential: From this ranking, the flow of measured under standard electrons inside the electron conditions (pH = 7; 25°C; 1M) transport chain from NADH to oxygen is predictable. 25  The electron-transport chain Complex I is the largest protein component of the inner mitochondrial membrane. It passes electrons from NADH to CoQ. The electrons from NADH are transferred first to FMN-, then iron-sulfur center- containing subunits of the complex, and finally to coenzyme Q. This electron transfer is also coupled with the extraction of protons from the mitochondrial matrix that are moved into the intermembrane space. 26 Complex II contains the dimeric citric acid cycle enzyme succinate dehydrogenase and three other small hydrophobic subunits. It passes electrons from FADH2 to CoQ. In the citric acid cycle, succinate dehydrogenase catalyzes the conversion of succinate into fumarate, with concomitant transfer of electrons and protons to the enzyme’s FAD-containing subunit to form FADH2. Thus, this complex allows the direct coupling of citric acid cycle with oxidative phosphorylation. The standard redox potential for electron transfer from succinate to CoQ is insufficient to provide the free energy to drive ATP synthesis. Coenzyme Q / Ubiquinone plays a central role in collecting electrons and protons from universal donors. It passes electrons from Complex I and II to Complex III. CoQ is a small, hydrophobic molecule that moves freely in the inner mitochondrial membrane. Complex III contains two b-cytochromes, one cytochrome c1, and a subunit with iron-sulfur center. It passes electrons from reduced CoQ to cytochrome c. 27 The electron transfer from CoQ to cytochrome poses a challenge. Coenzyme Q can donate two electrons but cytochrome c can only accept one electron. Complex III solves this problem with having two separate paths for the electrons that will lead to the production of two molecules of reduced cytochrome c. This process, called the Q cycle, provides also a mechanism to transport additional protons across the inner membrane of mitochondria. Cytochrome c is a peripheral membrane protein that is loosely bound to the outer surface of the inner mitochondrial membrane. It alternately binds to cytochrome c1 complex of Complex III and cytochrome c oxidase (Complex IV) and thereby functions to shuttle electrons between them. Complex IV acts a dimer, each monomer contains 13 subunits. In the functional core of Complex IV a series of carriers: copper centers and heme A prosthetic groups transfer the electrons. Cytochrome c oxidase catalyzes the one-electron oxidations of four consecutive reduced cytochrome c molecules and the concomitant four- electron reduction of one O2 molecule. Complex IV also transfers protons from the matrix to the intermembrane space. 28  The chemiosmotic hypothesis How does the electron transfer lead to ATP synthesis? The answer is given by the chemiosmotic hypothesis proposed by Peter Mitchell in 1961. The electron flow, through a series of carrier molecules and proteins, moves in a downhill exergonic direction, providing energy to power the uphill transport of protons across the inner mitochondrial membrane from the matrix to the intermembrane space. The free energy derived from the oxidation of fuel molecules, and ultimately from the electron transfer through the electron transport chain, is conserved as a transmembrane chemical potential called the proton motive force. 29 The electrochemical potential or proton motive force consists of two components: the membrane potential (charge difference, the matrix is more negative than the intermembrane space) and the H+ concentration difference (the matrix is more alkaline than the intermembrane space). (Electrical potential energy component + chemical potential energy component.) This gradient of protons is, in fact, an energy reservoir that is used to drive ATP synthesis. ATP is produced when the protons flow back into the mitochondrial matrix through the ATP synthase. The proton motive force can cover the energy requirement of various processes in the different organisms (heat, mechanical work, active transports), but cannot provide energy for biosynthetic processes. Biosynthetic processes require energy stored in the chemical bonds of ATP molecules. 30  The ATP synthase The F0 subunit of the enzyme is embedded in the mitochondrial membrane. It contains a ring of hydrophobic polypeptides that span the inner mitochondrial membrane, and a proton channel. The F1 subunit is located in the matrix. It contains chains that interact with the F0 subunit, and the so-called αβ-ring, which is the enzymatic core of the ATP synthase. Proton flow through the proton channel of the F0 subunit causes a rotation of the F0 subunit. The movement is transmitted onto the F1 subunit, which in turn, causes a conformational change of the β-chains. Different conformational states support the binding of ADP + Pi, trapping of these substrates, the conversion of ADP + Pi into ATP, and finally the release of ATP. 31 Under normal conditions, ATP synthesis requires an active electron transport. There is an obligatory coupling between the transport of electrons and the synthesis of ATP. This suggests that the regulation of ATP synthesis is based on the availability of fuel molecules. If the ADP/ATP ratio in the cell shifts in favor of ADP, this is a signal to increase oxidative phosphorylation. The role of the ADP level in determining the rate of oxidative phosphorylation is called respiratory control or acceptor control. 32