Citric Acid Cycle

Anmerkungen:

• 1. At times, fatty acid recovery 2. Provides intermediates for other reactions & pathways 3. Has a series of 8 reactions: Oxidizing acetyl group of acetyl-CoA to 2 molecules of CO2 & ocnserving energy in NADH & FADH2
• 4. "Central hub" of all metabolism

1. Output

   Anmerkungen:

   • 1. 1 complete cycle yields 2 CO2, 3 NADH, 1 FADH2. 1 "high energy" compound-GTP or ATP
   1. Oxidation

      Anmerkungen:

      • 1. Oxidation of acetyl coA to 2Co2 requries the transfer of 4 pairs of electrons. 2. Reduction of 3NAD+ to 3NADH accounts for 3 pairs 3. Reduction of FAD to FADH2 accounts for 4th pair 4. 11 ATP formed from these 4 electron pairs after eventual tranfer to O2
2. Net Reaction

   Anmerkungen:

   •  3NAD+ + FAD + GDP + P + acetyl-coA --> 3NADH + FADH2 + GTP + CoA + 2CO2
   • Oxaloacetate consumed in first and last step
   1. Transport

      Anmerkungen:

      • 1. TCA occurs in mitochondria 2. All substrates including NAD+ & GDP generated in or transported into mitochondria 3. All products consumed in or transported out of mitochondria
3. Purpose

   Anmerkungen:

   • 1. Reduces carbon structures down to CO2 2. Produces reducing equivalents used in electron transport & subsequently reoxidized by O2
   • 3. Central pathway for recovering energy from Carbs, fatty acids, amino acids that have been broken down to acetyl-coA
4. Input

   Anmerkungen:

   • 1. Pyruvate & NADH are final products of glycolysis 2. Pyruvate-H+ symport moves pyruvate and H+ into mitochondria

1. Pyruvate Dehydrogenase Complex

   Anmerkungen:

   • 1. Pyruvate is catalyzed by pyruvate dehydrogenase to Acetyl Co-A
   • 2. Group of noncovalently bound associated enzyme complexes
   1. Multienzyme complex

      Anmerkungen:

      • Advantages: 1. Increase reaction rate because complex minimizes distance 2. Side reactions decreased; channeling of metabolic intermediates between successive enzymes 3. Coordinately controlled
      1. 3 Enzymes

         Anmerkungen:

         • 1. Pyruvate dehydrogenase E1 2. Dihydrolipoyl transacetylase E2 3. Di hydrolipoyl dehydrogenase E3
         • 2. Core 60 units  (20 trimers) E2
      2. 5 Reactions
         1. 5 Cofactors

            Anmerkungen:

            • -Thiamine pyrophosphate (TPP), lipoamide, coA, FAD, NAD}
         2. 1st Rxn

            Anmerkungen:

            • 1. Pyruvate dehydrognease E1 requires cofactor TPP. Decarboxylates pyruvate to form hydroxyethyl TPP intermediate.
            • 2. TPP thiazolium ring can add carbonyl group and act as electron sink
         3. 2nd Rxn

            Anmerkungen:

            • 1. Hydroxyethyl group transferred to next enzyme dihydrolipoyl transacetylase E2. E2 contains a lipoamide group - lipoic acid linked via amide bond
            • 2. Reaction center is cyclic disulfide; reduced to yield dihydrolipoamide 3. Hydroxyethyl group derived from pyruvate attacks lipoamide disulfide with TPP eliminated
            • 4. Hydroxyethyl carbanion is oxidized to an acetyl group as the lipoamide disulfide is reduced 5. Lipoamide group on E2 is key to moving intermediate between E1 & E3
            1. Lipoamide group

               Anmerkungen:

               • 1. Acts as long teher swinging disulfide group from E1 where it pics up hydroxyethyl group to E2 active site where hydroxyethyl group transferred forming acetyl coA
               • 3. Swings from there to E3 where reduced disulfide is reoxidized
         4. 3rd Rxn

            Anmerkungen:

            • 1. E2 catalyzes transesterification reaction in which acetyl-group transferred to CoA yielding acetyl-CoA and dihydrolipoamide-E2
         5. 4th Rxn

            Anmerkungen:

            • 1. Acetyl-CoA formed 2. Must regenerate E2 lipoamide. E3 oxidizes & completes catalytic cycle of E2 3. Oxidized E3 contains reactive Cys-Cys disulfide group & tightly bound FAD
            • 4. Oxidation of dihydrolipoamdie is disulfide interchange reaction
         6. 5th Rxn

            Anmerkungen:

            • 1. Reduced E3 reoxidized. Sulfhydryl groups reoxidized by FAD funneling electrons to NAD+ yielding NADH
   2. Arsenic

      Anmerkungen:

      • 1. Inhibits lipoamide containing enzymes including pyruvate dehydrogenase complex and alpha-ketogluturate dehydrogenase 2. Stops respiration: TCA cycle
   3. Regulation of Pyruvate Dehydrogenase Complex
      1. 1st Regulatory System

         Anmerkungen:

         • 1. Product inhibition by NADH and acetyl CoA. Drives E2 & E3 backwards 2. Competitive inhibitors with NAD and CoA binding sites
         1. Conc. of NADH & Acetyl CoA

            Anmerkungen:

            • 1. High [NADH]/[NAD+] & [AcetylCo-A]/[CoA] maintains E2 in acetylated form. Ties up TPP on E1 and E2 cannot accept hydroxyethyl group
      2. 2nd Regulatory System
         1. Covalent Modificiation of E1
            1. Pyruvate Dehydrogenase Kinase
               1. Activated by NADH & acetyl CoA
                  1. Inactivates enzyme + ATP
            2. Pyruvate Dehydrogenase Phosphatase
               1. Activate enzyme
                  1. When glucose increases, promotes synthesis of acety-CoA & glycogen
      3. Other Regulators
         1. Inhibit pyruvate dehydrogenase

            Anmerkungen:

            • 1. Pyruvate, ADP 2. Ca+2 (also activates phosphatase) 3. no effect from cAMP

Anmerkungen:

• 1. Catalyzes condensation of acetyl-CoA and oxaloacetate 2. Point at which carbon atoms from carbs, fatty acids, and amino acids enter cycle
• 3. Ordered sequential. Oxaloacetate binds prior to acetyl-CoA
• Flux: varies with [substrate] Inhibited by Citrate & succinyl-CoA

1. Mechanism
   1. Oxaloacetate + Acetyl CoA
      1. Rate-limiting step and hydrogen bond

         Anmerkungen:

         • 1. Enol of acetyl-CoA generated in rate-limiting step when Asp 375 (a base) removes proton from methyl group 2. His 274 forms hydrogen bond with enolate oxygen
         1. Citryl-CoA + Citrate

            Anmerkungen:

            • 1. Formed in concerted acid-base catalyzed step 2. Citryl-CoA hydrolyzed to citrate and coA. 3. Free energy = -31.5 KJ/mol
      2. Acetyl-CoA binding site

         Anmerkungen:

         • 1. By x-ray studies, know that free enzyme is dimer in"open" form 2. 2 domains form a cleft containing oxaloacetate binding site
         • 3. Oxaloacetate binds smaller domain, rotates 18 degrees. Explains enzyme's ordered sequential kinetics.
         • 4. Rotation generates acetyl-CoA binding site seals oxaloacetate binding site, excluding water

Anmerkungen:

• 1. Catalyzes reversible isomerization of citrate to isocitrate with cis-aconitate as intermediate
• 2. Begins with dehydration. Proton & OH removed. 3. Citrate has 2 carboxymethyl groups attached to central C atom. Prochiral rather than chiral then becomes chiral
• Flux: near EQ

1. Citrate --> cis-Aconitate --> Isocitrate

   Anmerkungen:

   • 1. Aconitase contains iron-sulfur cluster. [4Fe-4S] coordinates -OH group of citrate to facilitate its elimination. Iron-sulfur clusters common in redox processes
   • 2. Rehydration of double bond of cis-aconitate to isocitrate

Anmerkungen:

• 1. NAD+ dependent E catalyzes oxidative decarboxylation of isocitrate to alpha-ketoglutarate
• 2. Inhibited by product NADH

1. 1st CO2 & NADH

   Anmerkungen:

   • 1. This CO2 began TCA as part of oxaloacetate and not acetyl CoA 2. Requires Mn+2 and Mg+2 cofactor & catalyzes oxidation of 2nd alcohol (isocitrate) to ketone (oxalosuccinate)
   • 3. Decarboxylation of carboxyl group Beta to ketone
   1. Isocitrate --> Oxalosuccinate --> Alpha-Ketoglutarate

Anmerkungen:

• 1. Inhibited by NADH & succinyl-CoA

1. 2nd CO2 & NADH

   Anmerkungen:

   • 1. CO2 entered as part of oxaloacetate rather than acetyl-CoA
2. Resembles pyruvate dehydrogenase multienzyme complex

1. "High-energy" cleavage

   Anmerkungen:

   • 1. Couples cleavage of "high energy" succinyl-CoA to synthesis of "high energy" GTP
   • 2. GTP normally synthesized from GDP + P. Energetically equivalent to ATP 3. Free energy = 0KJ/mol
   1. Passed to GTP
2. 3 Step Process

   Anmerkungen:

   • 1. Succinyl-CoA reacts with phosphate forming succinyl-phosphate and CoA
   • 2. Phosphoryl group transferred from succinyl phosphate to His residue on E releasing succinate
   • 3. Phosphoryl group on E transferred to GDP forming GTP
   1. 1 Acetyl Equivalent oxidized to

      Anmerkungen:

      • 2CO2, 2NADH, 1GTP

1. Dehydrogenation of Succinate to fumarate

   Anmerkungen:

   • 1. Stereo-specific dehydrogenation of S to F 2. E strongly inhibited by malonate. Ex. of competitive inhibitor
   • 3. 1. E contains FAD covalently linked 2. General biochem reaction oxidizes alkanes to alkenes
   1. Produces FADH2

      Anmerkungen:

      • 1. Reoxidized before next catalytic cycle 2. 2 electrons passed into electron transport
2. Only membrane-bound enzyme
   1. Funnels electrons directly into electron transport

1. Hydration of Double bond to form malate

   Anmerkungen:

   • 1. Hydration proceeds via cabanion transition state. OH addition occurs before H+ addition

1. Final step. Regeneration of oxaloacetate

   Anmerkungen:

   • 1. OH group of malate oxidized in NAD+ dependent reaction
   1. [Malate] high

      Anmerkungen:

      • 1. Delta G0 = +27 KJ/mol. 2. At EQ, [oxaloacetate] very low relative to [malate]
      1. Endergonic RXN
2. Citrate Synthase

   Anmerkungen:

   • 1. Highly exergonic 2. Free deltaG0 = -31.5KJ/mol because of cleave of thioester bond of citryl CoA
   1. Exergonic RXN

      Anmerkungen:

      • 1. Coupled RXNs allow citrate formation to be exergonic evan at low [oxaloacetate]. Keeps TCA working

1. Regulators

   Anmerkungen:

   • 1. Availability of substrates 2. Need for TCA cycle intermediates as biosynthetics precursors 3. Demand for ATP
2. Glycolysis Output
   1. 2 Pyruvate, 2NADH, 2 ATP

      Anmerkungen:

      • 1. 2 net ATP. (Produces 4ATP but 2ATP required as input)
      1. 2 Pyruvate converted to 2acetyl-CoA & 2NADH
         1. TCA cycle: 3NADH, 1 FADH2, 12 ATP, 1 CO2 per turn
            1. 24 ATP from TCA cycle
3. 1 Glucose Molecule
   1. Oxidative Phosphorylation (TCA cycle + pyruvate decarboxylation+ ETC?)
      1. 8NADH + 2 FADH2+2NADH=32ATP
         1. +4ATP from substrate-level phosphorylation of glycolysis & TCA
            1. 36 ATP
         2. 32 ATP

1. 3 enzymes likely control candidates for deltaG

   Anmerkungen:

   • 1. Citrate synthesis 2. Isocitrate dehydrogenase 3. Alpha-ketogluturate dehydrogenase
   1. Most Crucial Regulators

      Anmerkungen:

      • 1. Substrates: Acetyl-CoA & Oxaloacetate 2. Product: NADH
      1. Additional Regulators

         Anmerkungen:

         • 1. ADP: Allosteric activator of isocitrate dehydrogenase 2. ATP inhibits isocitrate dehydrogenase 3 Ca+2 activates pyruvate dehydrogenase phosphatase
         1. Calcium

            Anmerkungen:

            • 1. Stimulates muscle contractions + production of ATP to fuel muscle contractions
2. Heart Muscle

   Anmerkungen:

   • 1. Flux proportional to O2 consumption 2. Regulated by feedback mechanisms that coordinate NADH production with energy expenditure
3. Muscle

   Anmerkungen:

   • 1. Muscle workload & respiration rate increases. Mitochondrial [NADH] decreases. [Oxaloacetate[ increases. Stimulates citrate synthase

Anmerkungen:

• 1. Gluconeogenesis 2. Fatty Acid biosynthesis 3. Amino acid biosynthesis using oxaloacetate & Alpha-ketogluturate