14 Control of citric acid cycle

Dr.Vikram Raval

CONTROL OF CITRIC ACID CYCLE

Objectives

1. To understand basis of citric acid cycle (central metabolic hub)
2. Overview of citric acid cycle and enzymes of TCA cycle
3. Various factors affecting citric acid cycle and control points (regulation)
4. Significance of citric acid cycle
5. Anaplerotic reactions

To understand basis of citric acid cycle (central metabolic hub)

• The citric acid cycle is the central hub for metabolic pathways. It is popularly known as tricarboxylic acid cycle (TCA cycle) as well as Kreb’s cycle to honour the inventor of this central metabolic path point

For your information (FYI):

Sir Hans Adolf Krebs (1900-1981) born on August 25th at Hildesheim, Germany was biochemist and physician who pioneered the study of cellular respiration, and energy production. Globally he is acclaimed for his contributions towards 2 of the important biochemical reactions in the body, citric acid cycle and urea cycle. The former, being the central metabolic reaction for energy generation in cells, is often eponymously known as the “Krebs cycle”, fetched him a Nobel Prize in Physiology or Medicine in 1953. Hans Kreb and Hans Kornberg jointly discovered the glyoxylate cycle as well, which is a substitute of TCA cycle observed in plants, bacteria, protists, and fungi.

• The citric acid cycle is the common terminal point for all metabolic pathways to generate energy from fuel in form of biomolecules such as amino acids, fatty acids as well as carbohydrates

• In eukaryotes, TCA cycle reaction takes place in mitochondria instead glycolysis which occurs in cytoplasm while for prokaryotes both glycolysis and TCA occurs in cytoplasm

• Here in mitochondria, a series of oxidation and reduction reactions occur which results in oxidation of acetyl group of two carbon dioxide molecules while reducing the coenzymes ATP generation takes place. The reduced coenzymes can be reoxidized through electron transport chain clubbed with energy (ATP) generation reaction

Figure 1: Citric acid cycle: Central metabolic hub

Overview of citric acid cycle and enzymes of TCA cycle

• First a 4- carbon compound (oxaloacetate) fuses with a 2-carbon acetyl moiety to yield a 6-carbon tricarboxylic acid (citrate) through a condensation reaction (aldol condensation)

• Thereafter an oxidative decarboxylation reaction occurs that converts citrate into its isomeric form for removal of a molecule of carbon dioxide and formation of 5-carbon compound (α-ketoglutarate)

• A similar reaction occurs again that selectively converts 5-carbon compound (α-ketoglutarate) into a 4-carbon compound (succinate) by oxidatively decarboxylation

• Oxaloacetate is later being regenerated from 4-carbon compound succinate by series of reaction through formation of fumarate and malate

• 2-carbon atoms enter the cycle as an acetyl moiety while 2-carbon atoms leave the cycle in the form of 2 molecules of carbon dioxide

• Oxygen is required for the citric acid cycle indirectly in as much as it is the electron acceptor at the end of the electron transport chain, necessary to regenerate NAD+ and FAD

• 3 hydride ions are being shifted to 3 molecules of NAD+, while a pair of hydrogen atoms is being transferred to 1 molecule of FAD

• The chief function of the TCA cycle is the harnessing of energy-rich electrons from carbon fuel molecules

• TCA cycle thus provides precursor molecules like amino acids and other reducing agents like NADH, FADH2 that can be used in various other biochemical reactions

• The citric acid cycle, in conjunction with oxidative phosphorylation, provides the vast majority of energy used by aerobic cells in human beings, greater than 95%

• The above reactions are catalyzed by at least 8 different enzymes whereas the entry of acetyl moiety into the cycle is regulated by the action of a key enzyme called pyruvate dehydrogenase complex (PDHc)

• These are listed as under along with the substrate and product formed

Various factors affecting citric acid cycle control points (regulation)

• Like glycolysis, TCA cycle too can be regulated at two levels that is at the entry level of substrates and at certain key steps that are catalyzed by enzymes

• The chief control point of citric acid cycle is believed to be the point of entry of fuel molecule, the acetyl-co-A. Formation of this moiety from various carbohydrate sources acts as major regulatory checkpoint. The reaction is an enzyme catalyzed reaction and pyruvate dehydrogenase enzyme. The activity of this enzyme complex is easily inhibited by ATP, NADH, fatty acids and acetyl-Co-A

• The PDH complex can be regulated by

 i. Allosteric modification: PDHc is allosterically inhibited by acetyl-Co-A and NADH

while activated by non-acetylated Co-A (Co-A-SH) and NAD+

ii. Covalent modification: The PDHc activities are dependent on stages of phosphorylation modulated by specific kinase (PDH kinase) and dephosphorylation catalyzed by discrete phosphatase (PDH phosphatase)

• NADH and acetyl-Co-A act as activator while pyruvate, ADP, Co-A-SH, Ca2+ and Mg2+ act as inhibitor for PDH kinase. On the contrary, the PDH phosphatase, activity is boosted by Mg2+ and Ca2+

• Citrate synthase is allosterically inhibited by both ATP as well as long chain fatty acyl-Co-A. ATP, NADH and succinyl-Co-A also act as inhibitor while ADP plays a role of activato

• Isocitrate dehydrogenase activity is inhibited by NADH and ATP. In constrast it is allosterically activated by ADP, as it enhances affinity towards the substrate

• α-ketoglutarate dehydrogenase is inhibited by succinyl Co-A, NADH and ATP while activity is stimulated by Ca2+ ions

• The reactions of citric acid cycle are very well regulated. Majority of the above-mentioned enzymes can be regulated in various ways  as mentioned in the table below

• Succinate dehydrogenase activity is regulated concentration of oxaloacetate inside the mitochondrial matrix, which in-turn depends on malate dehydrogenase activity

• Malate dehydrogenase activity heavily depends on the [NADH]/[NAD+] ratio and is regulated by the same

Significance of citric acid cycle

• The citric acid cycle is the only a pathway for conversion and interchange of metabolites formed in a cell by various metabolic process of amino acids, fatty acids as well as carbohydrates

•  It mainly provides substrates for various deamination and transamination reactions of amino acid metabolism

• Since it plays a dual role both as a catalolic and an anabolic process, it is often called an amphibolic pathway

• Citric acid cycle plays a vital role in energy generation thereby involved in number of catalolic processes. The citric acid cycle is the usual but ultimate pathway for the oxidation of number of intermediates including carbohydrates, lipids, and proteins

• This is quite regular since glucose, fatty acids, and majority of amino acids are metabolized to form acetyl-Co-A or other intermediates of the TCA cycle

• The function of the citric acid cycle is said to be vital because it harnesses energy rich electrons from carbon skeleton. 1 acetate unit approximately produces 12 moles of ATP with each cyclic turn

• The TCA cycle is also regarded to be anabolic in nature as a number of intermediates are produced by it that are capable for biological synthesis/built-up of numerous cellular compounds

• Citric acid cycle generates intermediates that potentially are involved in gluconeogenesis. Since TCA intermediates forms oxaloacetate, this paves way for gurther decarboxylation and conversion mediated by phosphor-enol-pyruvate carboxy kinase to yield glucose (in liver/kidney)

• Citric acid cycle also plays major role in biosynthesis of certain essential and non-essential amino acids via reversible transamination reactions. Moreover it also avails the body with carbon skeleton necessary for biosynthesis of amino acids like alanine,aspartate, asparagine, glutamate, glutamine, lysine, methionine, theronine and isoleucine

• Citric acid cycle is also involved in biosynthesis of fatty acid via a key intermediate i.e. Acetyl-Co-A. The action of PDH converts pyruvate to Acetyl-Co-A, a major substrate for long-chain fatty acid biosynthesis. Acetyl-Co-A concentration is solely dependent on activity of a TCA cycle intermediate i.e. citrate

• Citric acid cycle is involved indirectly in synthesis of Heme/Hame moiety as Succinyl-co-A condenses with amino acid Glycine to form an α-amino-β-keto-adipic acid, a precursor for heme/haem biosynthesis

• TCA intermediates like glutamate and aspartate are precursors for purine and pyrimidine biosynthesis which in-turn is involved in formation of vital biomolecule of cell that is DNA

Anaplerotic reactions

• Anaplerosis here means restoring various intermediates of citric acid cycle that has been used in various reactions mentioned above in amphibolic processes of a cell

• Here the intermediates are reformed or refed into the cyclic pathway that had been previously extracted for various biosynthesis popularly called cataplerotic reactions

• As earlier mentioned TCA Cycle is a metabolic hub with hub of metabolism, with vital importance in both energy yielding and biosynthesis processes. Thus, it is critical for the cell to maintain intracellular concentrations of various intermediates

• Steady state (homeostasis) will only be maintained when the anaplerotic flux is balanced by cataplerotic flux of TCA cycle intermediates during cellular metabolism

• Below mentioned are few reaction that recharge the flux of intermediates used is various biosynthetic pathways:

i. Formation of oxaloacetate from pyruvate by simple carboxylation reaction in presence of pyruvate carboxylase

ii. Formation of oxaloacetate from malate by malate dehydrogenase [malate is

formed by malic enzyme that converts pyruvate into malate]

iii. Formation of oxaloacetate from aspartate by a transamination reaction

iv.Formation of alpha keto glutarate by transamination reaction of in presence of enzyme glutamate dehydrogenase

v.Formation of Succinyl-Co-A can be done in two ways either by oxidation of odd chain fatty acids or by methionine or isoleucine metabolism. Here first propionyl-Co-A is formed then methyl melonyl-Co-A and succinyl-Co-A

References

  Books

• Harper’sIllustrated Biochemistry by Robert K.. Murray, Daryl K. Granner, Peter A. Mayes, 26th Edition (2003)
• Lehninger’S Principle of Biochemistry David L. Nelson and Michael M. Cox,6th Edition (2008)
• Instant Notes: Biochemistry, 2nd Edition, B.D. Hames & N. M. Hooper (2005)
• Textbook of Biochemistry, 4th Edition Donald Voet, Judith G. Voet(2011)
• Biochemistry Berg, Tymoczko and Stryer 7th Edition (2010)

Web site

• http://themedicalbiochemistrypage.org/tca-cycle.html
• http://themedicalbiochemistrypage.org/tca-cycle.php
• http://www.rcsb.org/pdb/101/motm.do?momID=153
• http://library.med.utah.edu/NetBiochem/tabletit.htm#bioen