• Oxidize acetyl-CoA
• the 4 and 5C intermediates are used elsewhere for biosynthesis
• cell uses anaplerotic (replenishing) rxns to replace these intermediates
• [image]
• [image]

• acetyl-CoA donates acetyl group to oxaloacetate to begin cycle
• this forms 6C citrate
• which becomes isocitrate
• dehydrogenated, lose CO2, forming alpha ketoglutarate
• lose another CO2=succinate
• succinate then converted over three steps back into oxaloacetate

• Condensation of oxaloacetate with acety-CoA
• the hydrolysis of a high energy thiol intermediate makes rxn exergonic
• large - delta G is essential b/c concentration of oxaloacetate is very low
• CoA recycled back to PDH
• (picture of citrate formation)

• claisen condensation involving a thioester (acetylcoa) and ketone(oxaloacetate)
• (step 1 mechanism picture)
• oxaloacetate binds first (specifically oriented in the active site) to citrte synthase and opens binding site for acetyl coa

Step 2: citrate->cis-aconitate->isocitrate

• (add picture of step 2)
• aconitase can reversibly add water to ezyme bound cis aconitate, leading to citrate and iso citrate
• though small amount of isocitrate  at equilibrium, rxn is pulled to the right because isocitrate is rapidly consumed, lowering its steady state concentration

• (add picture of step 3)
• Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase forms alphaketoglutarate
• oxalosuccinate formed in active site as intermediate
• enol formed is stabilized by Mn++
• NAD(P)+ rxn occurs in mitochondrial matrix and cytosol and creates NAD(P)H, used for anabolism

Step 4: Oxidation of alpha-ketoglutarate to succinyl-CoA and CO2

• (add picture of step 4)
• another oxidative decarboxylation
• NAD+ is electron acceptor
• CoA is carrier of succinyl group
• oxidation energy of alpha-ketoglutarate conserved in forming thioester bond of succinyl CoA (and large -delta G)
• alpha-ketoglutarate dehydrogenase complex is similar to PDH complex: has E1, E2, E3 and TPP, lipoate, FAD, NAD and CoA. differs in that it binds a-ketoglutarate, not pyruvate

• (add step 5 pic)
• Succinyl coa has a thioester bond and the  negative std free energy of hydrolysis isused to drive synthesis of phosphoanhydride bond of ATP or GTP, resulting in -2.9 kJ/mol after coupling
• energy conserving reaction where His residue is phosphorylated in active site. this P is xfered to ADP/GDP->ATP/GTP
• formation of GTP/ATP at the expense of free energy released by a-ketoglutarate decarboxylation is a subtrate level phosphoryation
• GTP formed can ->ATP:    GTP+ADP-->GDP + ATP  delta G=0kJ/mol

• (add picture of step 6)
• enzyme succinate dehydrogenase is tightly bound to mitochondrial inner matrix (in euks)
• electrons pass from succinate through FAD to iron (now we're mentioning  oxidative phosphorylation) sulfur centers and then to chain of carriers in inner mitochondria membrane
• O2 is the final electon acceptor and generates 1.5 ATP/electron pair

•  
  • (Show step 7 picture)
• Reversible rxn catalyzed by fumarase, has carbanion T state
• fumarase is highly sterospecific and only binds the trans double bond of fumarate, not the cis (this isomer is called maleate)

• (step 8 picture)
• NAD linked L-malate dehydrogenase catalyzes the rxn of L-malate to oxaloacetate
• under standard conditions reaction lies far to the left but within cells reaction goes right b/c oxaloacetate is continually removed by the highly exergonic citrate synthase reaction (step1)
• this means [oxaloacetate] in cells is very low, forcing a favorability of oxaloacetate formation

• condensation of oxalo. + acetate expected to produce a citrate labeled in one of the two carboxyl groups because citrate is a symmetrical molecule
• half labled citrate molecules are expected to yeild a-ketoglutarate labeled in the gamma carboxyl group and half in alpha carboxyl group
• instead, only gamma is labeled, why? because enzyme it reacts with is assymetric
• this results in binding to enz. at three points in active site. This induced chirality of citrate due to enz. interation makes citrate prochiral

• (add energy conservation picture)
• two carbon acetyl group combines with oxaloacetate
• oxidation of isocitrate and a-ketoglutarate yeilds CO2
• oxidation energy conserved in reduction of 3 NAD+ and one FAD and production of one ATP/GTP
• four oxidation steps provide flow of electons to respiratory chain via NADH and FADH2

• Meaning it can serve in both catabolism and anabolism

(show massive anabolic pathway)

• plays a role in oxidative catabolism of fatty acids, carbs, and a. acids
• a-ketoglutarate is precursor to asp and glu (transamination)
• oxaloacetate-->glucose in gluconeogenesis
• succinyl coa-->porphyrin ring in heme grps

• as intermediates of the cycle are removed they are replenished by anapleurotic rxns
• (see figure on biosynthetic intermediate card; red arrows are anapleurotic)
• ex) reversible carboxylation of pyruvate to CO2 to make oxaloacetate. when cycle is defficient in oxalo.(or other intermediates), pyruvate is carboxylated to form more oxaloacetate

• Pyruvate carboxylase reaction requires vitamin biotin, which carries carbons in their most oxidized form, CO2
• carboxyl groups are activated in a  reaction that consumes ATP and joins CO2 to enzme bound biotin= activated CO2
• activated CO2 is then passed to acceptor (like pyruvate) in a carboxylation reaction
• pyruvate carboxyase has 4 subunits each containing biotin attached to a lys residue in the active site
• pyruvate carboxylation has two steps: first a carboxyl group derived from HCO3- is attached to biotin. Next the carboxyl group is transferred to pyruvate to form oxaloacetate.
• arm of biotin transfers activated carboxyl group from first active site to second (where ea/ step happens)
• lipoate, biotin, and pantothenate all become covalently attached to proteins by similar reactions and all can channel substrates (like biotin)
• (add pyruvate carboxylase mechanism)
  

• carbon flow into citric acid cycle is regulated at the conversion of pyruvate to acetyl-CoA
• also regulated at the entry of acetyl-CoA into the cycle
• availability of intermediates like fatty acids used for acetyl-CoA synthesis
• also by isocitrate dehydrogenase and a-ketoglutarate dehydrogenase rxns

• PDH is inhibited by ATP, acetyl-CoA, and NADH products. allosteric inhibition enhanced when long chain fatty acids are available (b/c--->acetyl-CoA)
• AMP, NAD+ and CoA activate PDH (accumulate when too little acetate flows into the cycle)
• also regulated by reversibly phosphorylateing on E1 ser residue (by two proteins)
• the kinase is allosterically activated by high [ATP] and E1 is phosphorylated, causing PDH rxn velocity to decline
• when [ATP] goes down, kinase activity goes down and the phosphatase removes the phosphoryl group from E1

• Three factors regulate the rate of flux through the cycle: substrate availability, product inhibition, and allosteric feedback inhibition of enzymes that catalyze early steps of the cycle
  
• (show 16-18 picture)
• the strongly exergonic steps catalyzed by citrate synthase,isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase can become rate limiting
• availability of oxaloacetate and acetyl-CoA for citrate synthase varies with rate of citrate formation
• NADH, product of isocitrate and a-ketoglutarate oxidation and when [NADH]/[NAD+] is high, both rxns are inhibited
• succinyl-CoA inhibits a-ketoglutarate dehydrogenase+citrate synthase
• citrate blocks citrate synthase
• end product ATP blocks isocitrate dehydrogenase and citrate synthase

• ADP allosterically activates citrate synthase
• in muscle, calcium (signal for contraction and increase in ATP) activates isocitrate dehydrogenase and a-ketoglutarate dehydrogenase as well as the PDH complex

• CAtalyze condensation reaction in which no nucleotide triphosphate (ATP/GTP) is required as an energy source

• Catalyze the condensations that do use ATP or another nucleotide triphosphate energy for the synthetic reaction

Transfer a phosphoryl group from a nucleotide triphosphate to an acceptor molecule. this is a phosphorylation.