TRICARBOXYLIC ACID CYCLE

TRICARBOXYLIC ACID CYCLE

Introduction: Also known as citric acid cycle or Krebs' cycle. It is the 2nd part of aerobic respiration. It occurs in the mitochondria. It is the final common pathway for the aerobic oxidation of carbohydrate, lipid, and protein. It also has a central role in gluconeogenesis, lipogenesis, and interconversion of amino acids.

 In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. Bacteria also use the TCA cycle to generate energy, but since they lack mitochondria, the reaction sequence is performed in the cytosol with the proton gradient for ATP production being across the plasma membrane rather than the inner membrane of the mitochondria. Theoretically there are several alternatives to the TCA cycle, however the TCA cycle appears to be the most efficient.

Formation of Acetyl CoA

Pyruvate produced by glycolysis enters the mitochondrion and is converted to acetyl CoA. Pyruvate (C3) --> acetyl CoA (C2) + CO2 NADH is produced from NAD+ + 2H (oxidation). This step must occur twice for each glucose molecule because each glucose molecule produces two pyruvate molecules in glycolysis. The two-carbon compound produced is attached to Coenzyme A to produce acetyl CoA.

Formation of Acetyl CoA

The enzyme involved is Pyruvate dehydrogenase.

Step 1:Formation of citrate

Acetyl CoA enters into mitochondria for the first reaction of Tricarboxylic acid cycle. Acetyl CoA participates in a condensation reaction with the four-carbon compound, oxaloacetate, to produce citrate. The reaction is catalyzed by citrate synthase. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, which is then hydrolyzed to citrate and CoA

 The hydrolysis of citryl CoA, a high-energy thioester intermediate, drives the overall reaction far in the direction of the synthesis of citrate.

Step 2: Isomerization of Citrate

Citrate is isomerized to isocitrate by the enzyme aconitase. It is a two step reaction. 1) dehydration to cis-aconitate 2) rehydration to isocitrate.

Step 3:Oxidation of Isocitrate to Ketoglutarate and CO2

It is the first oxidative decarboxylation step. Isocitrate is converted to a-Ketoglutarate. The reaction is catalyzed by the enzyme Isocitrate dehydrogenase. The reaction involves dehydrogenation to Oxalosuccinate, an unstable intermediate which spontaneously decarboxylates to give aKetoglutarate

 The decarboxylation requires Mg2+ or Mn2+ ions. In addition to decarboxylation, this step produces an NADH cofactor.

Step 4: Oxidation of -Ketoglutarate to Succinyl-CoA and CO2

Second oxidative decarboxylation step. -ketoglutarate is converted to succinyl-CoA and CO2 by the action of the -ketoglutarate dehydrogenase complex. NAD is the electron acceptor and CoA the carrier of the succinyl group

Step 5: Conversion of Succinyl-CoA to Succinate

Succinyl-CoA is a high potential energy molecule. The energy stored in this molecule is used to form a high energy phosphate bond in a Guanine nucleotide diphosphate (GDP) molecule. The enzyme that catalyzes this reversible reaction is the succinyl-CoA synthetase

Step 6: Flavin-Dependent Dehydrogenation

The succinate formed from succinyl-CoA is oxidized to fumarate by succinate dehydrogenase. The hydrogen acceptor is FAD since the free energy change is insufficient to reduce NAD+.

Step 7: Hydration of Fumarate to Malate

The reversible hydration of fumarate to L-malate is catalyzed by fumarase. This enzyme is highly stereospecific; it catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate (the cis isomer of fumarate). In the reverse direction (from L-malate to fumarate), fumarase is equally stereospecific: D-malate is not a substrate

Step 8: Oxidation of Malate to Oxaloacetate

Malate is oxidized to form oxaloacetate. This reaction is catalyzed by malate dehydrogenase, and NAD+ is the hydrogen acceptor.

 The equilibrium of this reaction lies far to the left under standard thermodynamic conditions, but in intact cells oxaloacetate is continually removed by the highly exergonic citrate synthase reaction. Thus the concentration of oxaloacetate in the cell remains low , pulling the malate dehydrogenase reaction toward the formation of oxaloacetate.

Summary of Reactions
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O

2 CO2 + COA-SH + 3 NADH + 3 H+ + FADH2 +GTP

Regulation of the citric acid cycle

The citric acid cycle is regulated primarily by the concentration of ATP and NADH. The major control points are the enzymes isocitrate dehydrogenase and -ketoglutarate dehydrogenase. Isocitrate dehydrogenase is inhibited by high [ATP] and [NADPH]. Inhibition by ATP is relieved by high [ADP] which enhances the affinity of the enzyme for the substrate.

 -ketoglutarate dehydrogenase is inhibited by its products ie succinyl CoA and NADH. It is also inhibited by a high energy charge ie when [ATP] is high. Thus , the rate of the cycle is regulated by the level of ATP within the cell.

Citric acid cycle in E.coli

In E.coli the citric acid cycle switches to a branched, non-cyclic mode under anaerobic conditions. The cycle is 'broken' at the alpha-ketoglutarate dehydrogenase step and takes a reverse path to succinyl-CoA starting from oxaloacetate via malate/aspartate, fumarate, and succinate. The branched citric acid 'cycle' mode provides biosynthetic precursors succinyl-CoA and alphaketoglutarate.

 Oxaloacetate itself is used up in this reaction and needs to be replenished from the combination of two acetate units produced in the complete oxidation of pyruvate. This is performed by the glyoxylate cycle in bacteria (and plants; in the organelles called glyoxysomes).

Glyoxylate cycle
The glyoxylate cycle is a variation of the tricarboxylic acid cycle. It is an anabolic pathway occurring in plants, bacteria, protists, fungi and several microorganisms, such as E. coli and yeast. In this cycle, isocitrate is split by isocitrate lyase into succinate and glyoxylate. The latter can be linked with acetyl-CoA to form malate and CoA in a reaction catalyzed by malate synthase.

Major metabolic pathways converging on the TCA cycle

Several catabolic pathways converge on the TCA cycle. Proteins are broken down into amino acids which can be converted into acetyl-CoA and enter the TCA cycle. Triglycerides are hydrolyzed fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde3-phosphate by gluconeogenesis.

 In many tissues, especially heart tissue, fatty acids are broken down through a process known as beta oxidation. This results in formation of acetyl-CoA, which can be used in the citric acid cycle. Beta oxidation of fatty acids with an odd number of methylene groups produces propionyl CoA, which is then converted into succinyl-CoA and fed into the citric acid cycle.

Nucleotide Metabolism
Nucleotides play a variety of important roles in all cells. They are the activated precursors of DNA and RNA. ATP, an adenine nucleotide, is a universal currency of energy in biological systems. GTP is an essential carrier of chemical energy. Adenine nucleotides are components of the coenzymes NAD+, NADP+, FMN, FAD and Coenzyme A.

 UDP-Glucose in Glycogen synthesis and CDPdiacylglycerol in Phosphoglyceride synthesis are the nucleotide derivatives that act as activated intermediates. Cyclic AMP is a ubiquitous mediator for the action of many hormones. All cells can synthesize nucleotides from simple building blocks (de novo synthesis) or by the recycling of pre-formed bases (Salvage pathway).

 Nucleotides are phosphate esters of pentoses in which a nitrogenous base is linked to C1 of the sugar residue. A nucleotide without the phosphate group is known as a nucleoside. The major purine components of nucleic acids are adenine and guanine residues. The major pyrimidine residues are those of Cytosine, Uracil and Thymine. Pyrimidines are bound to ribose through N 1 atoms.

Synthesis of purine ribonucleotides

IMP is synthesized from ribose 5-phosphate. There are 11 reactions in the formation of IMP. IMP is converted to GMP and AMP with the help of ATP and GTP respectively. Nucleoside monophosphates are converted to nucleoside diphosphates by base specific monophosphate kinases.

 Purine nucleotide synthesis is regulated by feedback inhibitor AMP, GMP and IMP. An important regulatory factor is the availability of PRPP. Salvage pathway for purines is observed in RBC and the brain. Free purines are salvaged by APRTase and HGPRTase enzymes

Synthesis of pyrimidine ribonucleotides

Pyrimidine ring is synthesized as free pyrimidine and then it is incorporated into the nucleotide. Six reactions are involved in the synthesis of UMP. UDP and UTP are synthesized from UMP with the help of ATP. CTP is formed by adding an amino group from glutamine. Pyrimidine can also be salvaged using PRPP.

 In orotic aciduria, excretion of large amount of orotic acid is observed. It results from the deficiency of either orotate phospho ribosyl transferase or OMP decarboxylase.

Formation of deoxyribonucleotides
Ribonucleotide reductase catalyzes the synthesis of deoxyribonucleotide. The reductant is NADPH. Thioredoxin transfers electrons from NADPH for reduction of 2-OH of ribose. dTMP is formed by thymidylate synthase by methylation of deoxy uridine monophosphate.