ENERGY YIELD 6 CO2 5 Total = 12 ATP 4 Citrate Isocitrate CH3C O ~ S-CoA Citrate Isocitrate a-ketoglutarate Succinyl-CoA Succinate Fumarate Malate Oxaloacetate CO2 6 5 4 ENERGY YIELD NADH + H+ 3 ATP 3 ATP 1 ATP 2 ATP NADH + H+ 3 ATP FADH2 Total = 12 ATP NADH + H+ GTP

Prochirality Ogston’s 3 Point Attachment Theory Aconitase CH2-COO HO-C-COO Prochirality Symmetrical CH2COO OH C C HO CH2COO OOC COO CH2COO CH2-COO Ogston’s 3 Point Attachment Theory

Ogston’s Three-point Attachment Theory of Aconitase Test this yourself Middle Index Right Hand Thumb Only your right hand will fit the pattern. Therefore, only one of the isomers of citrate will fit on the enzyme surface in the correct orientation

.. Glyoxysomes Glyoxylate Cycle Citrate Isocitrate a-ketoglutarate CH3C O ~ S-CoA Glyoxylate Cycle (germinating plant seeds) Glyoxysomes (plant organelles) Starch H-C-COO HO-C-COO H .. Malate Synthase CO2 Citrate Isocitrate a-ketoglutarate Succinyl-CoA Succinate Fumarate Malate Oxaloacetate CHO COO Isocitrate Lyase Glyoxylate All 6 carbons are preserved Bypass COO- CH2 Mitochondria

Glyoxysome Mitochondria P 625 Aspartate Aspartate Oxaloacetate -Ketoglutarate -Ketoglutarate Glutamate Glutamate Oxaloacetate Oxaloacetate NAD+ NADH Acetyl-CoA HS-CoA Malate Citrate Fumarate Isocitrate Isocitrate lyase FAD FADH2 Glyoxylate Succinate Succinate Malate synthase Acetyl-CoA HS-CoA Malate NAD+ NADH P 625 OAA Gluconeogenesis Starch

Summary of Reactions

6 4 2 1 ATP 9 ATPs 2 ATPs

Where do we get all that energy? 1. How energetic is citrate? 6 Carbons = 3 Cycle Turns = 3 x 12 ATP per cycle = 36 ATPs 2. How energetic is oxaloacetate 4 Carbons to Citrate = 2 Cycle turns = 24 ATPs 3. How energetic is malate 4 Carbons = 2 Cycle turns + 1 NADH = 27 ATPs

Regulation of the Kreb’s Cycle Pyruvate Dehydrogenase complex Pyruvate + TPP  Acetal-TPP + CO2 Acetal-TPP + S-S  Ac-S ^ SH + TPP Ac-S ^ SH + HS-CoA  AcS-CoA + HS ^ SH HS ^ SH + FAD  S-S + FADH2 FADH2 + NAD+  FAD + NADH + H+ Pyruvate + HS-CoA + NAD+  Acetyl-CoA + NADH + H+ Regulators-Activators Regulators- Inhibitors and AMP Fatty acids and ATP

Key Regulatory Points: 1. Pyruvate dehydrogenase Complex Inhibited by NADH and Acetyl-CoA NADH [NAD+] Acetyl-CoA HS-CoA High NADH means that the cell is experiencing a surplus of oxidative substrates and should not produce more. Carbon flow should be redirected towards synthesis. High Acetyl-CoA means that carbon flow into the Krebs cycle is abundant and should be shut down and rechanneled towards biosynthesis

Mechanism: 1. Competitive Inhibition Text p621 1. Competitive Inhibition NADH and acetyl-CoA reverse the pyruvate dehydrogenase reaction by competing with NAD+ and HS-CoA 2. Covalent Modification (second level regulation) E-1 subunits of PDH complex is subject to phosphorylation TPP FAD 1 2 3 Epinephrine Glucagon E1-OH E1-OPO3 H2O HPO4= ATP ADP PDH kinase phosphatase Active Inactive Cyclic-AMP protein kinase Insulin ATP

All regulatory enzymes occur in the first half of the cycle Regulation of Cycle Enzymes Go’ (kJ/mol) Enzyme Citrate Synthase -31.5 Aconitase ~5 Isocitrate dehydrogenase -21 -Ketoglutarate dehydrogenase -33 Succinyl-CoA Synthase -2.1 Succinate dehydrogenase +6 Fumarase -3.4 Malate dehydrogenase +29.7 All regulatory enzymes occur in the first half of the cycle

Regulation of the Citric Acid Cycle Primary modes: 1. Substrate availability (key enzymes are subsaturated) 2. Product inhibition Allostery is not a primary mode 3. Feedback inhibition (competitive) Key regulators: 1. Acetyl-CoA (controls citrate synthase) 2. OAA (controls citrate synthase, regulated by NADH) 3. NADH (controls citrate synthase, isocitrate dehydrogenase 4. Calcium (stimulates NADH production)

Controls NADH and is controlled by NADH Equilibria to Consider O2 consumption NADH oxidation ATP production Controls NADH and is controlled by NADH Tightly coupled: affect one is to affect all Malate + NAD+ OAA + NADH A working muscle will increase respiration and oxidize NADH. This stimulates OAA synthesis which stimulates citrate synthase and isocitrate dehydrogenase reactions.

Citrate Malate + NAD+ OAA + NADH Substrate limited K = [OAA][NADH] -Kg dehydrogenase Isocitrate dehydrogenase K [Malate][NAD+] [OAA][NADH] Respiration (O2) Respiration Increases

P 621 Pyruvate Dehydrogenase Citrate Synthase No Regulation Isocitrate -Ketoglutarate Dehydrogenase