UCL Biochemistry And Molecular Biology Lecture 19 PDF

BENG0004 Biochemistry and Molecular Biology Emily Kostas Lecture 19 The TCA cycle (citric acid cycle, Krebs cycle) The major catabolic pathways Glycolysis The pentose...

BENG0004 Biochemistry and Molecular Biology Emily Kostas Lecture 19 The TCA cycle (citric acid cycle, Krebs cycle) The major catabolic pathways Glycolysis The pentose phosphate pathway b-oxidation  The TCA cycle (citric acid cycle, Krebs cycle) In 1937 Sir Hans Krebs based at the University of Sheffield summarised the known work on a cycle of reactions that included citric acid and that could explain the complete oxidation of sugars. This was based on many scientists’ previous work through the 1930s and some of the enzymes were identified and characterised by Albert Szent-Györgyi who had worked on fumarate and vitamin C. So the name Krebs cycle is from Sir Hans Krebs and the citric acid cycle from the first compound made when acetyl CoA joins the cycle and the tricarboxylic acid cycle from the three carboxylates on citrate. or Mammalian cell major catabolic pathways  The TCA cycle (citric acid cycle, Krebs cycle) This cycle takes acetyl CoA generated by the breakdown of sugars, fats (lipids) and proteins to generate GTP and reduced cofactors NADH and FADH2 Overall 1 molecule of acetyl CoA are converted to 2 molecules of CO2 and 3 NADH, 1 FADH2 and 1 GTP The NADH and FADH2 are oxidised at the electron transport chain which we will look at in another lecture. At the end of the glycolytic pathway pyruvate is the product. This must be converted into acetyl CoA for entry into the TCA cycle, using pyruvate dehydrogenase. CO2 is lost and a molecule of NADH is made.  The Pyruvate dehydrogenases complex Pyruvate Dehydrogenase catalyzes oxidative decarboxylation of pyruvate, to form acetyl-CoA. It is found in the cytosol of bacteria e.g. E. coli and in the matrix of mitochondria. It is a very important enzyme as it converts the products of sugar metabolism to the acetyl CoA needed for the TCA cycle. There are three subunits E1, E2, and E3 and in E. coli there are 24 E1, 24 E2 and 12 E3 proteins. In mammalian cells there are 60 copies of E2, 30 copies of E1 and 12 copies of E3 and 12 copies of a binding protein that links E2 to E3. E1 is itself a tetramer of subunits a2b2. So the mammalian enzyme is a complex of 294 protein subunits!  The Pyruvate dehydrogenases complex The E2E3 complex Bacillus stereothermophilus PDH The E2 inner core, 24 subunits In eukaryotic cells the TCA cycle enzymes are all within the mitochondrial matrix. In E.coli all the enzymes are in the cytosol (cytoplasm). In eukaryotic cells pyruvate is generated outside the mitochondria from glycolysis and enters the mitochondria via a pyruvate/H+ symporter (this means the pyruvate and H+ enter together) and consumes energy. PDH in the mitochondrial matrix generates acetyl CoA Acetyl CoA from b-oxidation is generated within the mitochondrial matrix. Pyruvate + H+ Pyruvate + CoA SH  The entry of acetyl CoA into the TCA cycle TCA cycle reaction 1 Citrate is formed from the irreversible condensation of acetyl CoA and oxaloacetate – catalyzed by citrate synthase. This reaction has a large negative ΔG so the reaction has an equilibrium far over to the right. TCA cycle reaction 2 Citrate is too stable so it is isomerised by aconitase to D-isocitrate. Cis-aconitate is an intermediate. TCA cycle reaction 3 Isocitrate dehydrogenase – catalyses an oxidative decarboxylation of isocitrate to a-ketoglutarate. The a-ketoglutarate (also known as 2-oxoglutarate) is a link to nitrogen metabolism. The amino acid glutamate is derived from a-ketoglutarate by replacing the ketone group with an ammonium ion. Transaminases can also feed the TCA cycle with a-ketoglutarate by removal of the NH2 from glutamate. TCA cycle reaction 4 A very large enzyme complex called a-ketoglutarate dehydrogenase removes CO2 , CoA is added and NADH is formed. TCA cycle reaction 5 Succinyl CoA synthetase – the bond between succinate and CoA is particularly unstable and the energy it holds can be used to make GTP. This is converted to ATP in mitochondria by the enzyme nucleoside diphosphate kinase. In E. coli the SCS enzyme can generate either ATP or GTP. TCA cycle reaction 5, contd. Succinyl CoA synthetase – during catalysis the phosphate is first transferred to the succinate preserving the energy that was in the succinyl CoA ester bond. Then the phosphate is transferred to a histidine on the enzyme and finally the phosphate is transferred to GDP (or ADP) to make GTP. TCA cycle reaction 6 Succinate dehydrogenase – this is an enzyme complex that has part of it in the inner membrane of the mitochondrion. It links its citric acid cycle task directly to the electron transport chain. It first extracts hydrogen atoms from succinate, transferring them to the carrier FAD. With the help of several iron- sulfur clusters and a heme, these are then transferred to the mobile carrier ubiquinone, for transport to cytochrome bc1.  The cofactor FAD, FADH2 Flavin adenine dinucleotide, can carry 2 H+ and 2e- (two protons and two electrons) in its fully reduced form The flavin can exist in three forms. FADH2 is the Flavin most fully reduced. +2H+ 2e- Ribitol FADH 2 Adenine The FADH2 is tightly bound to the enzyme and the electrons captured in the FADH2 are transferred to a lipid soluble electron carrier called ubiquinone that always stays in the membrane. Succinate dehydrogenase with its membrane insertion helices Ubiquinone Ubiquinol TCA cycle reaction 7 Fumarase – water is added across the carbon=carbon double bond to form L- malate. In mammalian cells the enzyme is found both in the mitochondrion and in the cytoplasm, where it is involved in amino acid catabolism and the urea cycle. TCA cycle reaction 8 Malate dehydrogenase –The final step of the citric acid cycle recreates oxaloacetate, transferring electrons to NADH in the process. Malate dehydrogenase is found in both the mitochondrion and the cytoplasm.  Precursors for biosynthetic pathways The TCA cycle also has a role in producing precursors for biosynthetic pathways. These include: 1) Synthesis of fatty acids from citrate 2) Amino acid synthesis following the transamination of α-ketoglutarate 3) Synthesis of purines and pyrimidine nucleotides from α-ketoglutarate and oxaloacetate 4) Oxaloacetate can be converted to glucose by gluconeogenesis 5) Succinyl CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups Biochemistry Berg et al, 8th edition. TCA cycle – pages495-497, 500-510.