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1,2,3,5,6,8,10,11,16,20,21 Citric Acid Cycle Reactions

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1,2,3,5,6,8,10,11,16,20,21 Citric Acid Cycle Reactions Overview of the citric acid cycle, AKA the krebs cycle AKA tricarboxylic acid AKA TCA cycle Suggested problems from the end of chapter 19: 1,2,3,5,6,8,10,11,16,20,21 Glycogen is broken down into glucose. The reactions of glycolysis result in pyruvate, which is then fed into the citric acid cycle in the form of acetyl CoA. The products of the citric acid cycles are 2 CO2, 3 NADH, 1 FADH2, and 1 ATP or GTP. After pyruvate is generated, it is transported into the mitochondrion, an organelle that contains the citric acid cycle enzymes and the oxidative phosphorylation enzymes. In E. coli, where there are neither mitochondria nor other organelles, these enzymes also seem to be concentrated in certain regions in the cell. Citric acid cycle reactions Overall, there are 8 reactions that result in oxidation of the metabolic fuel. This results in reduction of NAD+ and FAD. NADH and FADH2 will transfer their electrons to oxygen during oxidative phosphorylation. •In 1936 Carl Martius and Franz Knoop showed that citrate can be formed non-enzymaticly from Oxaloacetate and pyruvate. •In 1937 Hans Krebs used this information for biochemical experiments that resulted in his suggestion that citrate is processed in an ongoing circle, into which pyruvate is “fed.” •In 1951 it was shown that it was acetyl Coenzyme-A that condenses with oxaloacetate to form citrate. 1 The pre-citric acid reaction- pyruvate dehydrogenase Pyruvate dehydrogenase is a multi-subunit complex, containing three enzymes that associate non-covalently and catalyze 5 reaction. The enzymes are: (E1) pyruvate dehydrogenase (E2) dihydrolipoyl transacetylase (E3) dihydrolipoyl dehydrogenase What are the advantages for arranging enzymes in complexes? E. coli pyruvate dehydrogenase complex The E. coli complex is a particle of about 300 Å in diameter. The mass is ~4.6 million Daltons (g/mol). There are 60 polypeptides per particle. The complex contains 24 E2 proteins which are arranged in a cube and form the so-called core complex. 24 E1 proteins and 12 E3 proteins are arranged around the core. 2 The E. coli and human pyruvate dehydrogenase complexes share structural features “Core” complex, E2 (dihydrolipoyl transacetylase) proteins associate as trimers at the corners of the cube. E1 (pyruvate dehydrogenase) dimers (in yellow/orange) associate with the E2 trimers at the edges of the cube. E3 (dihydrolipoyl dehydrogenase) dimers (light blue) associate at the six faces of the cube. Pyruvate dehydrogenase catalyzes five reactions 3 Pyruvate dehydrogenase reactions Reaction 1 involves decarboxylation of pyruvate in a reaction that depends on the co-enzyme thiamine pyrophosphate (TPP). The mechanism resembles that of the pyruvate decarboxylase reaction, except the product here is hydroxyethyl TPP:E1. Thiamine pyrophosphate H3C O O O O Aminopyrimidine -O C O H C C O P O P O- -C ring 2 H H O C O + C N 2 2 C N - S O O- R Thiazolium R ring H H3C N NH2 H3C Hydroxyethyl-TPP:E1 R' R N+ S - H O C CH3 Pyruvate dehydrogenase reactions In reaction 2 the hydroxyethyl group is transferred to dihydrolipoyl transacetylase (E2). The E2 co-enzyme is a lipoamide group. The lipoamide is attached to the enzyme via an amide linkage to a lysine side chain. The lipoamide can interconvert with the dihydrolipoamide form. 4 Pyruvate dehydrogenase reactions In reaction 2 of pyruvate dehydrogenase the hydroxyethyl group attacks the lipoamide disulfide. The hydroxyethyl carbanion is oxidized to an acetyl group and the lipoamide group is reduced. Pyruvate dehydrogenase reactions Reaction 3. E2 catalyzes a transesterification reaction, in which the acetyl group is transferred to CoA. Acetyl-CoA will be used in the citric acid cycle, but the dihydrolipoamide group must be oxidized to regenerate the E2 enzyme. 5 Thioesters Thioesters are considered “primitive” high-energy compounds. They are found in the central metabolic pathways of all known organisms. Thioesters were, presumably, more common in the pre- biotic world than phosphate compounds. Phosphates are less abundant in non-living systems than thioesters. In metabolic pathways the thioester appears as a part of acetyl-coenzyme A (CoA). This molecule is an intermediate in carbohydrate, fatty acid, and amino acid catabolism. ∆Gº’ of acetyl-CoA hydrolysis is -31.5 kJ/mol. Pyruvate dehydrogenase reactions Reaction 4. E3, dihydrolipoyl dehydrogenase, is used to regenerate the lipoamide group. A reactive cys-cys disulfide group is used together with a non-covalent, tightly bound FAD. Reaction 5. Re-generation of E3. The sulfhydryl groups are oxidized by FAD, and FAD is oxidized by NAD+, which results in the production of NADH. 6 Pyruvate dehydrogenase reactions The E2 protein on the inside of the structure must “channel” intermediates from the E1 and E3 enzymes on the outside of the structure. This apparently is achieved due to the flexibility and length of the lipoyllysyl arm. This arm (shown on right) may swing between E1 and E3. In terms of the reactions that are catalyzed, this arm can be thought of as a “tether” that brings the reactive disulfide group to the active site of E1 (where it reacts to accept the hydroxyethyl group), then swings to the active site of E2 (where CoA accepts the acetyl group), and finally to the active site of E3 (where the disulfide group is re-oxidized). Support for this idea is the finding that the lipoyllysyl arm is attached to the E2 protein via an alanine-proline rich region that seems to adopt multiple conformations, meaning that it is flexible. If this idea is correct, a single lipoyllysyl arm can reach numerous E3 active sites. How would you test the idea that a flexible linkage near the lipoamide group is essential for the activity of pyruvate dehydrogenase? Structure of E2 of pyruvate dehydrogenase The E2 catalytic domain from the bacterium Azotobacter vinelandii is shown. The 24 E2 proteins are arranged in trimers, each trimer occupying the corner of a cube. The edge of the cube is ~125 Å. Substrates and co-factors must fit in the spaces between the E2 proteins and within the interior cavity of the cube. Only 4 “corners” are shown. Acetyl CoA must diffuse into its binding site from the internal space of the cube. 7 Arsenic effects on pyruvate dehydrogenase S OH HS -O As + -O As + 2 H2O OH HS S R R Arserite Dihydropiloamide 3- •Arsenite compounds (AsO3 ) and organic arsenicals (R—As=O) inhibit aerobic respiration by reacting with lipoamide-containing enzymes (pyruvate dehydrogenase is just one of these enzymes). Asrenicals also can affect bacterial enzymes, and apparently at lower doses, which is the reason for the treatment of syphilis and trypanosomiasis with arsenicals before the advent of modern antibiotics. Side effects were severe. •Arsenic may be consumed unwittingly. Napoleon, for example, may have suffered a chronic poisoning. Many wallpaper dyes at that time contained arsenic, and mold growing on the walls in damp weather (for example, on Elba) can convert the arsenic to volatile form, which can then be ingested. •Fowler ’s tonic, which many people took to remedy various ailments, contained 10 mg/ml arsenite. Reaction 1. Citric Synthase - proposed catalytic mechanism The reaction involves condensation of acetyl CoA and oxaloacetate into citric acid. His-274, histidine-320, and aspartate-375 have been implicated in general acid - general base catalysis. 8 Reaction 1. Citric Synthase - proposed substrate binding mechanism oxaloacetate There is no binding affinity detected for acetyl-CoA in the absence of oxaloacetate. Oxaloacetate is bound in the presence or absence of acetyl-CoA. In the presence of oxaloacetate alone there is no catalytic reaction, but the above conformational change is observed. What substrate binding mechanism is supported by these observations? Reaction 2. Aconitase The enzyme contains a 4Fe-4S iron-sulfur cluster. The enzyme is unusual in that it does not catalyze a redox reaction, as most iron-sulfur enzymes do. In the second stage of this reaction, addition of water across the the double bond can, in principle, result in 4 stereoisomers. The enzyme is stereospecific in the sense that only one isocitrate stereoisomer is produced by the enzyme. 9 Reaction 2. Aconitase The iron not bound to a cysteine sulfur coordinates the C3 OH and COO- groups of citrate. This iron accepts an electron from the hydroxyl group, which then becomes the leaving group. Reaction 3. Isocitrate dehydrogenase + This reaction produces the first CO2 and NADH of the citric acid cycle. The enzyme is NAD - dependent •To achieve the decarboxylation reaction, magnesium or manganese can be utilized by the enzyme. •The CO2 that is released originated from oxaloacetate (not acetyl-CoA). •Specific mutations in this enzyme help elucidate the catalytic mechanism because these mutations did not completely destroy catalysis, rather these mutations slowed some of the steps in the pathway such that intermediates accumulated. For example, the presence of oxalosuccinate was not observed until mutations in lysine-230 and tyrosine-160 were made. These mutations act as kinetic “bottlenecks” such that reactions involving the intermediates are slowed down. 10 Reaction 4. α-ketoglutarate dehydrogenase This reaction resembles the pyruvate dehydrogenase multi-enzyme complex reaction, down to the identity of the E3 dihydrolipoyl dehydrogenase. As expected, the mechanisms are, most likely, the same, and the product is the high energy thioester, in this case, succinyl CoA. Reaction 5. Succinyl CoA synthetase Succinyl-CoA + ADP + Pi ↔ ATP + succinate + CoASH This enzyme couples the free energy released from hydrolysis of the high energy thioester succinyl CoA to the synthesis of a high energy NTP. GTP is generated in mammalian cells, whereas ATP is generated in bacteria and plant cells. From an energetic standpoint, these reactions essentially are the same because GTP and ATP can be interconverted rapidly through the action of nucleoside diphosphate kinase.
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