Biological oxidation II. The Cytric acid cycle

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Elfrieda Wiggins
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1 Biological oxidation II The Cytric acid cycle

2 Outline The Cytric acid cycle (TCA tricarboxylic acid) Central role of Acetyl-CoA Regulation of the TCA cycle Anaplerotic reactions The Glyoxylate cycle Localization of CAC in the cell

3 Bridging Step - Pyruvate Decarboxylase Entry - Citrate Synthase All the Other Steps Intermediates for Other Pathways

4 The TCA Cycle Citric Acid Cycle, Krebs Cycle Pyruvate (actually acetate) from glycolysis is degraded to CO 2 Some ATP is produced More NADH is made NADH goes on to make more ATP in electron transport and oxidative phosphorylation

5 The Cytric Acid Cycle Tricarboxylic Acid Cycle Krebs Cycle

6 The Chemical Logic of TCA Understand this! TCA seems like a complicated way to oxidize acetate units to CO 2 But normal ways to cleave C-C bonds and oxidize don't work for CO 2 : cleavage between Cs and to a carbonyl an -cleavage of an -hydroxyketone A better way to cleave acetate... Better to condense acetate with oxaloacetate and carry out a -cleavage - TCA combines this with oxidation to form CO 2, regenerate oxaloacetate and capture all the energy as NADH and ATP!

7 Entry into the TCA Cycle Pyruvate dehydrogenase and citrate synthase Pyruvate is oxidatively decarboxylated to form acetyl-coa Pyruvate dehydrogenase uses TPP, CoASH, lipoic acid, FAD and NAD + NADH and succinyl-coa are allosteric inhibitors

8 Aconitase Isomerization of Citrate to Isocitrate Citrate is a poor substrate for oxidation So aconitase isomerizes citrate to yield isocitrate which has a secondary -OH, which can be oxidized Aconitase uses an iron-sulfur cluster

9 Isomerization of Citrate to Isocitrate

10 Isocitrate Dehydrogenase Oxidative decarboxylation of isocitrate to yield - ketoglutarate Classic NAD + chemistry (hydride removal) followed by a decarboxylation Isocitrate dehydrogenase is a link to the electron transport pathway because it makes NADH

and mechanistically Five coenzymes used - TPP, CoASH, Lipoic acid,

11 -Ketoglutarate Dehydrogenase A second oxidative decarboxylation This enzyme is nearly identical to pyruvate dehydrogenase - structurally and mechanistically Five coenzymes used - TPP, CoASH, Lipoic acid, NAD +, FAD You know the mechanism if you remember pyruvate dehydrogenase

12 Succinyl-CoA Synthetase A substrate-level phosphorylation A nucleoside triphosphate is made Its synthesis is driven by hydrolysis of a CoA ester

13 Succinate Dehydrogenase An oxidation involving FAD Mechanism involves hydride removal by FAD and a deprotonation This enzyme is actually part of the electron transport pathway in the inner mitochondrial membrane The electrons transferred from succinate to FAD (to form FADH 2 ) are passed directly to ubiquinone (UQ) in the electron transport pathway

15 Fumarase Hydration across the double bond trans-addition of the elements of water across the double bond Possible mechanisms are shown in Fig. The actual mechanism is not known for certain

17 Malate Dehydrogenase An NAD + -dependent oxidation The carbon that gets oxidized is the one that received the - OH in the previous reaction This and the previous two reactions form a reaction triad that we will see over and over!

18 CA Cycle Summary One acetate through the cycle produces two CO 2, one ATP, four reduced coenzymes The Fate of Carbon in TCA Carboxyl C of acetate turns to CO 2 only in the second turn of the cycle (following entry of acetate) Methyl C of acetate survives two cycles completely, but half of what's left exits the cycle on each turn after that.

21 Intermediates for Biosynthesis The TCA cycle provides several of these -Ketoglutarate is transaminated to make glutamate, which can be used to make purine nucleotides, Arg and Pro Succinyl-CoA can be used to make porphyrins Fumarate and oxaloacetate can be used to make several amino acids and also pyrimidine nucleotides

23 Intermediates for Biosynthesis The TCA cycle provides several of these Note (Fig.) that mitochondrial citrate can be exported to be a cytoplasmic source of acetyl-coa and oxaloacetate

24 The Anaplerotic Reactions The "filling up" reactions PEP carboxylase - converts PEP to oxaloacetate Pyruvate carboxylase - converts pyruvate to oxaloacetate Malic enzyme converts pyruvate into malate PEP carboxykinase - could have been an anaplerotic reaction, but it goes the wrong way! CO 2 binds weakly to the enzyme, but oxaloacetate binds tightly, so the reaction goes the wrong way.

26 The Reductive TCA Cycle Reversed TCA cycle The TCA cycle running backward could assimilate CO 2 This may have been the first metabolic pathway Energy to drive it? Maybe reaction of FeS with H 2 S to form FeS 2 (iron pyrite) iron pyrite, which was plentiful in ancient times, and which is an ancient version of iron-sulfur clusters!

27 Regulation of the TCA Cycle Again, 3 reactions are the key sites Citrate synthase - ATP, NADH and succinyl-coa inhibit Isocitrate dehydrogenase - ATP inhibits, ADP and NAD + activate -Ketoglutarate dehydrogenase - NADH and succinyl-coa inhibit, AMP activates Also note pyruvate dehydrogenase: ATP, NADH, acetyl-coa inhibit, NAD +, CoA activate

29 The Glyoxylate Cycle A variant of TCA for plants and bacteria Acetate-based growth - net synthesis of carbohydrates and other intermediates from acetate - is not possible with TCA Glyoxylate cycle offers a solution for plants and some bacteria and algae The CO 2 -evolving steps are bypassed and an extra acetate is utilized Isocitrate lyase and malate synthase are the shortcircuiting enzymes

31 Glyoxylate Cycle II Isocitrate lyase produces glyoxylate and succinate Malate synthase does a Claisen condensation of acetyl-coa and the aldehyde group of glyoxylate - classic CoA chemistry! The glyoxylate cycle helps plants grow in the dark! Glyoxysomes borrow three reactions from mitochondria: succinate to oxaloacetate

Citric Acid Cycle: Central Role in Catabolism. Entry of Pyruvate into the TCA cycle

Citric Acid Cycle: Central Role in Catabolism Stage II of catabolism involves the conversion of carbohydrates, fats and aminoacids into acetylcoa In aerobic organisms, citric acid cycle makes up the final