The TCA tricarboxylic acid citric acid and krebs Cycle Eight Enzymes Purpose Oxidation of Fuel Formation of o Anabolic precursors o GTP 1 o NADH 3 o FADH2 1 Key reactions Regulation Three primary modes of regulation Availibility of substrate increase in substrate will drive reactions forward Product inhibition Competitive feedback by downstream intermediates Start and end with a four carbon molecule oxaloacetate bring in two carbons in the form of an acetyl group to form a 6 carbon then to oxidative decarboxylations o each will yield NADH and CO2 o ending with 4 carbons then form GTP or ATP depending on the tissue o also FADH2 and NADH The four carbon oxaloacetate brings in two carbons from acetyl group A condensation reaction that produces the six carbon molecule citrate Citrate undergoes an isomerization Water is going to leave then come back again forming Isocitrate Next the first oxidative decarboxylation Produces alpha ketoglutarate Redox reaction so NAD is needed to accept electrons Yielding NADH and CO2 and a proton The second oxidative decarboxylation Produces a four carbon Succinyl CoA NADH and a proton CO2 Substrate level phosphorylation occurs next ATP or GTP is produced and CoA Use GDP or ADP and inorganic phosphate Left with 4C Succinate Oxidation Reduction Oxidize succinate with FAD as the carrier ending up with FADH2 Left with 4C Fumarate Hydration of Fumarate is next Add water to form 4C Malate Oxidation Reduction of Malate Use NAD Left with 4c Oxaloacetate Produces NADH and proton For every turn 3 NADh 1 FADH2 1 GTP or ATP reducing equivalents used in ETC to make ATP Reaction 1 Enzyme Citrate Synthase Condensation reaction forming 6C molecule Formation of Citrate from Oxaloacetate and Acetyl CoA o 4C Oxaloacetate 2C Acetyl CoA yields Citryl CoA intermediate to 6C Citrate and CoA Roles of Citrate in Metaboism Mitochondria o Intermediate in the TCA cycle When theses levels build up goes to cyto Signal that theres a lot of energy around Cytoplasm o Activator of Fatty Acid Synthesis o Precursor of Cytoplasmic AcetylCoA Used to make fatty acids o Inhibiton of PFK1 No need for more AcetylCoA Citrate Synthase Dimer each monomer has a small and large domain Operates by sequential ordered reaction Oxaloacetate binds first and causes the small subunit to rotate o Forms Acetyl CoA binding pocket Brings closer the residues that are necessary for the condensation reaction Only active when both substrates are available o Induced fit Regulation of enzyme o Negative ATP NADH Succinyl CoA Reaction Two Enzyme Aconitase Isomerization in Two Steps o Dehydrations of citrates to yield a C C cis Aconitate intermediate o Stereospecific rehydration of the double bond yields Isocitrate Go from a tertiary alc to a secondary alc Reaction Three Enzyme Isocitrate Dehydrogenase o Oxidation reduction reaction NADH H produced Forms an unstable beta ketoacid intermediate oxalosuccinate o Decarboxylation reaction CO2 alpha ketoglutarate produced First produced NADH and CO2 Rate Limiting step Regulation o Positive ADP and NAD o Neg NADH direct comp with NAD and ATP Reaction Four Enzyme alpha ketoglutarate dehydrogenase complex A lot like pyruvate dehydrogenase complex Alpha keto glutarate NAD CoA yields succinyl CoA CO2 and NADH From now on 4C Alpha Ketoglutarate dehydrogenase A lot like PDC 3 enzymes E1 E2 and E3 5 cofactors o Tpp o Lipoamide o FAD prosthetic group o NAD o CoA Products Succinyl CoA NADh and CO2 Regulation o Pos AMP o Neg Succinyl CoA NADH Reaction 5 Enzyme succinyl CoA synthetase Named for reverse reaction Substrate level phosphorylation of GDP Production of GTP or ATP Succinyl Coa Pi GDP yield Succinate CoA GTP Reaction 6 Oxidation Reduction reaction o Using covalently attached FAD as cofactor o Electrons transferred to iron sulfur clusters in enzyme and ultimately O2 by the way of other members of ETC FADH2 produced 4C succinate yields Fumarate Enzyme succinate dehydrogenase o Flavin ring is covalently attached o Imbedded in the inner mito membrane o Part of the ETC Regulation o Negative FADh2 FAD ratio o High ratios inhibit Reaction 7 Hydration across the double bond sets up the next reaction Fumarate yields malate Enzyme Fumarase o Adds water in trans across DB to yield L Malate o Made possible by Fe S complex in enzyme Malate is used in gluconeogensis Reaction 8 Last oxidation reduction reaction Regenerates OAA NADH produced Malate yields OAA and NADH plus Proton Enzyme Malate Dehydrogenase Regulation o Negative NADH NAD Rxn must be able to reverse o Drive forward by removing product OAA Four Major points of Regulation PDC o Pos NAD CoA o Neg Acetyl CoA NADH ATP Citrate Synthase o Neg Succinyl CoA NADH and ATP IsoCitrate DH o Pos NAD ADP o Neg NADH ATP AKG dehydrogenase complex o Pos AMP o Neg Succinyl CoA NADH Individual reactions can be regulated at the level of products and reactants Le Chateliers Principle Things made in TCA are used for other things as well Citrate o Make fatty acids and sterols Alpha ketoglutarate o Make glutamate o Other amino acids o Purine nucleotides Succinyl CoA o Make prophyrins necessary for heme and chlorophyll OAA o o o Makes amino acid Aspartate Other AA Purines and pyrimidines Anapleroic Reactions Filling up Preventing shit from running out What would happen to the TCA cycle if succinyl CoA were continually removed to make heme for hemoglobin and the only input into the cycle was acetyl CoA We would soon run out of OAA Both citric acid cycle and glycolysis would slow and then the citric acid cycle would stop Mammals cannot use acetyl CoA for net conversion into oxaloacetate We lack the enzymes necessary for this to occur Acetyl CoA must enter the TCA cycle by way of condensation with oxaloacetate to form citrate and there is loss of two moles of CO2 for every acetyl entry to the cycle Transporting Reducing Equivalents into the Mito Matrix Oxidative phosphorylation overview ETC Integral membrane electron carries Mobile electron carrier Following Electrons through the ETC How do reducing equivalents get from the cyto into the mito matrix Hand of electrons from one carrier to another Cyto carrier to mito carrier Carriers themselves do not cross the membrane Glycerol 3 phosphate shuttle o NADH cyto and Mito E FAD prosthetic group that is bound into an enzyme o To NAD cyto and EFADH2 Mito Malate aspartate shuttle o NADH cyto and NAD mito o To NAD cyto and NADH mito Glycerol 3 phosphate Shuttle Sits on cytoplasmic face facing intermembrane space