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The Citric Acid Cycle II and the Pentose Phosphate Pathway 4/22/2003 The Citric acid cycle Overall reaction 3NAD+ + FAD + GDP + Pi + acetyl-CoA 3NADH + FADH + GTP + CoA + 2CO2 Overview 24 E2 subunits 24 E1 orange a and b together 12 E3 Red EM based image of the core E2 from yeast pyruvate dh Domain structure of dihydrolipoyl transacetylase E2 Acetyl reaction center transferes though the E2 dihydrolipoyl coenzyme repeats O CH3 S O HS S E2 O S E2 S S SH E2 S E2 S E2 CH3 S CH3 O S HS E2 SH E2 S S CH3 S E2 Citrate Synthase O HO CH2 C O O + H3C HO C O O HO CH2 HO C CH2 HO O O OH SCoA Induced fit needs binding of oxaloacetate before Acetyl CoA can bind. O CoAS C OH CoAS CH3 CH2 Acetyl-CoA Proposed intermediate OH O CoAS CH2 CoAS C CH3 Acetonly CoA (ground-state analog) C CH2 O Carboxymethyl-CoA (transition state analog) Aconitase O HO CH2 HO CH2 O O Citrate O HO CH2 O CH C CH2 HO O HO O HC OH OH CH HO HO O Cis-Aconitate HO C H OH O Isocitrate The double bond is placed on the Pro-R arm NAD+- Dependent Isocitrate dehydrogenase NAD+ NADH a-Ketoglutarate dehydrogenase O HO HO NAD+ CO2 CH2 CH2 H2C C HO O O O CH2 NADH CoAS O This enzyme is just like pyruvate dehydrogenase, a multi enzyme complex that is specific for longer CoA derivatives Succinyl-CoA Synthetase or succinate thiokinase Succinate dehydrogenase HO O CH CH2 O + 2e- + 2H+ CH CH2 HO O HO HO O The FAD on the enzyme itself is reduced Succinate dehydrogenase is the only membrane bound enzyme in the citrate cycle O H3CO CH3 Succ dh--FADH2 + CH2 H3CO n O Ubiquinone or Coenzyme Q n = 6-10 CH3 Oxidized form OH H3CO CH3 CH2 n H3CO OH CH3 Reduced form Fumarase Malate dehydrogenase O HO NADH H2 C H C O HO H2 C OH C O NAD+ HO O HO O Regulation of the citric acid cycle Standard free energy changes in the citric acid cycle Reaction 1 2 3 4 5 6 7 8 Enzyme Citrate synthase Aconitase Isocitrate dh a-KG dh Succinyl-CoA synthase Succinate dh Fumarase Malate dh DG' -31.5 ~5 -21 -33 -20.1 +6 -3.4 +29.7 DG' Negative ~0 Negative Negative ~0 ~0 ~0 ~0 The points of regulation of the cycle Citric acid cycle intermediates are always in flux A single molecule of glucose can potentially yield ~38 molecules of ATP Phosphopentose pathway Produces NADPH and ribose-5-phosphate NADH and NADPH although chemically similar they are not metabolically exchangeable. Many anabolic pathways require the reducing power of NADPH for synthesis including Fatty acid synthesis and the synthesis of cholesterol. 3G-6-P + 6NADP+ + 3H2O 3CO2 + 2F6P + GAP 6NADPH + 6H+ The pathway consists of three parts 1. Oxidative reactions: 3G-6-P + 6NADP+ + 3H2O 3Ribulose-5-PO4 6NADPH + 3CO2 + 2. Isomerization and epimerization reactions: 3Ribulose-5-PO4 Ribose -5-PO4 + 2Xylulose-5-PO4 3. A series of C-C bond cleavage and formations: Ribose-5-PO4 + 2Xyluose-5-PO4 2F-6-P + GAP Glucose-6 phosphate dehydrogenase Phosphogluconate dehydrogenase Ribulose-5-PO4 isomerase Two enzymes control the rearrangement of carbon skeletons which result in the production of Glyceraldehyde-3-phosphate and Fructose-6-phosphate. Transketolase transfers C2 units: TPP requiring enzyme like pyruvate dehydrogenase Transaldolase transfers C3 units: uses a shiffs base with an active lysine group Transketolase requires TPP The transition of carbon skeletons in the Phosphopentose pathway The pentose pathway control The need for NADPH is controlled by glucose dehydrogenase, however, when ribose -5phosphate is needed (DNA and RNA synthesis) it can be made from the reverse of the transaldolase and transketolase reactions from Fructose-6-PO4 and GAP NADPH is needed for glutathione reductase Reduced glutathione is needed for glutathione peroxidase, which destroy hydrogen peroxide and organic peroxides. This enzyme requires selenium as a cofactor. O H3+N CH CH2 CH2 C O NH COO - CH C NH CH2 COO - O CH2 2 S H3+N S COO H3+N CH - COO - CH2 CH2 CH2 C O NH CH CH CH2 CH2 C O NH CH CH2 SH C O NH CH2 COO - C NH CH2 COO - Glutathione keeps proteins with reduced sulfhydryls SH from oxidizing to R P-SH + P’-SH + O2 S S R’ P-S-S-P’ + H2O P-S-S-P’ G-SH P-SH + G-S-S-P G-SH G-S-S-G + HS-P Glutathione reductase contains FAD Reaction of glutathione with peroxides 2GSH + RA-O-O-H G-S-S-H + ROH + H2O A steady supply of glutathione is required for erythrocyte integrity ~ 400,000,000 individuals are deficient in glucose dehydrogenase! Without a fully functioning glucose dehydrogenase, glutathione concentrations Hemolytic Anemia can occur if certain drugs are used. Primaquine, an antimalarial drug is problematic with individuals with glucose dehydrogenase deficiencies CH3 NH CH CH2 CH2 CH2 NH 2 N H3CO Primaquine Similar effects are seen when people eat Fava beans. Fava beans stimulate peroxide formation and the demand for NADPH can not be met. Mature red blood cells lack a nucleus and the ability to make new proteins and membranes. Damage cannot be repaired so cells lyse. A defective G-6-P dh confers a selective advantage on individuals living where malaria is endemic. However, only heterozygotic females are resistant to malaria, not males. Plasmodium falciparum can adopt to a cell with decreased levels of phosphopentose products. This enzyme is in the X chromosome and females with two x chromosomes produce half good and half bad blood cells. Plasmodium cannot adapt to the G-6-P dh deficiency if it is sporadic or random.