Catabolism: the third stage

Intermediary Oxidative Metabolism The TCA Cycle or citric acid cycle or Krebs Cycle

Stages of catabolism  1 st stage: Large molecules in food are broken down into smaller units. Preparation stage without capture of energy. Proteins -> amino acids, Polysaccharides -> monosaccharides (glucose,...) Fats -> glycerol, fatty acids.  2 nd stage: Molecules are degraded to simple units that play a central role in metabolism. Most of them are converted into the acetyl unit of acetyl CoA. Some ATP is generated in this anaerobic stage, but amount is small compared with 3 rd stage.  3 rd stage: ATP is produced from the complete oxidation of the acetyl unit of acetyl CoA. Acetyl CoA brings acetyl units into the citric acid cycle, where they are completely oxidized to CO 2. Four pairs of electrons are transferred (three to NAD + and one to FAD) for each acetyl group that is oxidized. Then, a proton gradient is generated as electrons flow from the reduced forms of these carriers to O 2, and this gradient is used to synthesize ATP.

Third stage  Anaerobic generation of ATP from glucose via glycolysis is inefficient: Endproducts still contain energy that could be released by further oxidation.  The addition of an oxygen requiring stage (stage 3, oxidative metabolism) resulted in a more powerful means of extracting energy from food molecules.  The third stage of catabolism consists of the TCA cycle (or citric acid cycle or Krebs cycle) and the process of oxidative phosphorylation.  In citric acid cycle, acetyl groups from acetyl CoA are oxidized to produce CO 2 and NADH  In oxidative phosphorylation, NADH reacts with O 2 to produce ATP and H 2 O via a complicated series of steps involving mitochondrial electron transport.

From pyruvate to acetyl-CoA  Pyruvate is transported into the mitochondrial matrix by a pyruvate/OH - antiport.  In the matrix, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex to form acetyl CoA.  Acetyl CoA is then fed into the TCA cycle.

Other sources of Acetyl CoA

Beta oxidation of fatty acids Each FA molecule undergoes an initial activation step to become a fatty acyl-CoA – this is energized by hydrolysis of an ATP to AMP After activation, a repeating cycle of 4 reactions splits off acetyl Co-A until the end of the fatty acid is reached

The activation step

Fatty acids: β-oxidation  Fatty acids in cytosol are bound to CoA.  Transport into mitochondria  1 st oxidation at β C and transformation of FAD to FADH 2.  Hydration of double bond.  Oxidation and transformation of NAD + to NADH + H +.  Acetyl CoA splits off and rest of chain is bound to another CoA.  … until fatty acid is at its end.  Special cases are unsaturated fatty acids and fatty acids with odd numbers of C atoms.  Fatty acid oxidation takes also place in peroxisomes, but no ATP generation.  Acetyl-CoA back to cytosol (synthesis)

The beta oxidation spiral

Mitochondria do beta-oxidation Beta oxidation takes place inside mitochondria – so without mitochondria a tissue cannot metabolize fat for energy.

Ketone bodies and ketoacidosis High rates of fat oxidation tend to leave us with an overload of acetylCoA, which the liver converts to acetate, acetone, acetoacetate, and beta-OH butyrate. These are the so-called ketone bodies that appear in the blood, sweat and breath of individuals that are starving, or suffering from untreated diabetes mellitus. Some of them are acids, so their appearance in the blood causes ketoacidosis. Some of them are smelly, so they can be detected by a practitioner without the use of chemical analysis.

Odd vs even-numbered fatty acids Since fatty acids are taken apart (and also assembled) in 2-C pieces, some cells find it harder to deal with odd-numbered fatty acids – they are unusual in mammals but common in plants and marine organisms. The tail-end of an odd-numbered fatty acid turns out to be 3-C propionyl-CoA, which is decarboxylated in a multistep process to form pyruvate. So, we can eat oysters, after all.

Amino acids: protein is broken down to amino acids  Proteins are digested to amino acids that are delivered to the cells.

Amino acids: transamination and deamination  Transamination: an amino acid transfers its amino group to an α-keto acid. The amino acid becomes an α-keto acid and the α-keto acid becomes an amino acid:  In this way amino groups are collected in a few types of amino acids (often glutamate).  Oxidative deamination: an amino acid (often glutamate) is oxidized and deaminated with liberation of ammonium:

Amino acids: carbon skeletons enter catabolic pathways  The carbon skeletons of amino acids enter at different points into catabolic pathways.

TCA cycle: an overview  Primary function of TCA cycle is to oxidize acetyl groups that enter the cycle as acetyl CoA molecules (coming from pyruvate and fatty acids)  4-C compound condenses with 2-C acetyl unit to yield 6-C tricarboxylic acid  6-C compound is oxidatively decarboxylated to yield a 5-C compound  5-C compound is oxidatively decarboxylated to yield a 4-C compound  In subsequent reactions another NADH is produced as well as 1 FADH 2 and a high energy phosphate (GTP)  The electron carriers (NADH and FADH 2 ) yield 9 ATP when they are oxidized by O 2 in the electron transport chain.

Feeding acetyl CoA into the cycle  Enzyme: citrate synthase  CoA-SH is liberated to be again used for transfers

Citrate is isomerized  Enzyme: aconitase  For later oxidative decarboxylation the tertiary OH-group is not at the right place  Dehydration results in cis-Aconitate  Hydration results in Isocitrate with the OH-group at C 2.

Oxidative decarboxylation I  Enzyme: Isocitrate dehydrogenase

Oxidative decarboxylation II  Enzyme:  -ketoglutarate dehydrogenase  Reaction resembles oxidative decarboxylation of pyruvate

Generation of GTP  Enzyme: Succinyl CoA synthetase  Cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate, usually GDP.  This is a substrate-level phosphorylation  Phosphorylating GDP is equavalent energetically to phosphorylating ADP  GTP is not just ATP in another form - it is involved in signal transduction – a large number of intracellular signals are GTP-binding proteins

Regeneration of oxaloacetate  A methylene group (CH 2 ) is converted into a carbonyl group (C = O) in three steps: an oxidation a hydration a second oxidation  Oxaloacetate is regenerated for another round of the cycle  More energy is extracted in the form of FADH 2 and NADH.

Summary of TCA cycle (again)  2 CO 2 split off  3 NADH + H + and 1 FADH 2 generated  1 GTP generated  No oxygen involved, but TCA cycle is only active in the presence of oxygen! Why?

Summary of TCA cycle  Almost all reactions have negative ΔG.