3/9/151Biochemistry401Lecture 20 The TCA cycle: Eight ReactionsPurpose: Acquisition of Energy from FuelsProduction of Building blocks (Anabolic precursors)Oxidation, Oxidative Decarboxylations Formation of:NADH (3)FADH2 (1)GTP (1)Key ReactionsKey Regulated Steps (3 major points of regulation)These Notes are Color Coded: • Enzymes!• Inhibitors • Positive effectors • Important Anabolic Products Key thoughts regarding regulation: There are primarily 3 modes of regulation: 1. Availability of substrate 2. Product inhibition 3. Competitive feedback by downstream intermediates TCA Cycle: Tricarbocxylic Acid Cycle Citric Acid Cycle Krebs Cycle3/9/1523/9/1533/9/1543/9/1551. Oxaloacetate + Acetyl CoA + H2O ⇌ Citrate + CoA _____________________________________________________________________ 2. Citrate ⇌ Isocitrate _____________________________________________________________________ 3. Isocitrate + NAD+ ⇌ α-Ketoglutarate + CO2 + NADH + H+ _______________________________________________________________________________________________________ 4. α-Ketoglutarate + NAD+ + CoA ⇌ Succinyl CoA + CO2 + NADH + H+ _______________________________________________________________________________________________________ 5. Succinyl CoA + GDP (or ADP) + Pi ⇌ Succinate + CoA + GTP (or ATP) _______________________________________________________________________________________________________ 6. Succinate + FAD ⇌ Fumarate + FADH2 _______________________________________________________________________________________________________ 7. Fumarate + H2O ⇌ Malate _______________________________________________________________________________________________________ 8. Malate + NAD+⇌ Oxaloacetate + NADH + H+ _______________________________________________________________________________________________________ Can you match the type of reaction? (some letters are used more than once) !A. Redox!B. Oxidative decarboxylation!C. Isomerization !D. Hydration!E. Substrate level phosphorylation!F. Condensation!3/9/1561. Oxaloacetate + Acetyl CoA + H2O ⇌ _____________________________________________________________________________________________________________ 2. Citrate ⇌ _____________________________________________________________________________________________________________ 3. Isocitrate + NAD+ ⇌ _____________________________________________________________________________________________________________ 4. α-Ketoglutarate + NAD+ + CoA ⇌ ______________________________________________________________________________________________________________ 5. Succinyl CoA + GDP (or ADP) + Pi ⇌ _________________________________________________________________________________________________________________ 6. Succinate + FAD ⇌ _______________________________________________________________________________________________________________ 7. Fumarate + H2O ⇌ _______________________________________________________________________________________________________________ 8. Malate + NAD+⇌ _______________________________________________________________________________________________________________ Can you name the products? !Reaction 1 Enzyme : Citrate Synthase !Condensation Reaction forming 6C molecule Formation of Citrate from Oxaloacetate and Acetyl CoA intermediate 4C + 2C = 6C Condensation, driven by CoA hydrolysis3/9/157Reaction 1 Enzyme : Citrate Synthase !Condensation Reaction forming 6C molecule Formation of Citrate from Oxaloacetate and Acetyl CoA intermediate 4C + 2C = 6C Regulation: Negative: ATP, NADH, Succinyl CoA Sequential ordered reaction OAA binds first, and causes the small subunit of Citrate Synthase to rotate, this forms the Acetyl CoA binding pocket. This brings closer the residues that are necessary for the condensation reaction between OAA and Acetyl CoA to occur. Dimer - Each monomer has a small and large domain Example of induced fit Cytoplasm Mitochondria High Citrate Levels Indicate a Well Fed State3/9/158Reaction 2 Enzyme: Aconitase !Isomerization in 2 steps: A) Dehydration of Citrate to yield a C=C, cis-Aconitate B) Stereospecific Rehydration of the double bond yields Isocitrate Pro-chiral Plane of symmetry Chiral Reaction 2: Isomerization in 2 parts Why? Setting up for the first Redox Reaction Isomerization in 2 steps: Dehydration followed by re-hydration. Puts the hydroxyl group in better position for oxidation and CO2 release in the next step. (Oxidative Decarboxylation) Enzyme: Aconitase!Reaction 3 Enzyme: Isocitrate Dehydrogenase A) Oxidation - reduction reaction; NADH + H+ produced B) Decarboxylation reaction, CO2 + α-ketoglutarate produced First reaction in which CO2 is produced. NADH is also produced and will be used to make ATP. Unstable β-Ketoacid intermediate 6C - 1C = 5C3/9/159Regulation - !1) 4 major points of regulation !a) PDC ! NAD+, CoA Acetyl CoA, NADH, ATP!b) Citrate Synthase Succinyl CoA, NADH, ATP!c) Isocitrate DH -! NAD+, ADP NADH, ATP !!d) AKG DH - ! AMP Succinyl CoA, NADH!2) Individual reactions can be regulated at the level of products and reactants (Le Chatelier’s Principle)!Reaction 3: Isocitrate Dehydrogenase!The first of 4 oxidation - reduction reactions. CO2 release (First of 2) NADH produced Rate limiting step Regulation: Positive: ADP, NAD+ Negative: NADH (direct competition with NAD+) ATP Reaction 3 Enzyme: Isocitrate Dehydrogenase!ADP decreases KM of the enzyme, Isocitrate Dehydrogenase for its substrates.3/9/1510Addition of CoA and release of CO2. The enzymes are similar to those in Pyruvate Dehydrogenase Complex, E1 and E2 are homologous to those in PDH, and E3 is identical to PDH E3. Together, these enzymes make up the α-ketoglutarate Dehydrogenase complex. The second NADH and CO2 are produced. From now on, 4C. Reaction 4 “4th enzyme” is an enzyme complex :!α- Ketoglutarate Dehydrogenase Complex!α- Ketoglutarate Dehydrogenase Complex!This enzyme is a complex, like the Pyruvate Dehydrogenase Complex. E1 and E2 are similar, E3 is identical to PDC Regulation: Positive: AMPNegative: Succinyl CoA, NADH!Reaction 4 : Production of: Products: • 2nd NADH • 2nd (and final) CO2 • Succinyl CoA used for Heme synthesis Addition of CoA: Sets up the production of GTP in the next reaction. Reaction 5: Succinyl CoA
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