13 Glyoxylate cycle

Dr.Vikram Raval

epgp books

   

 

 

 

 

Glyoxylate cycle

 

Objectives

  1. To understand the biochemistry of Glyoxylate cycle.
  2. To understand the role of Glyoxylate cycle in gluconeogenesis.
  3. To understand relationship between the Glyoxylate cycle and TCA cycle.
  4. To study the function of the glyoxylate cycle in various organisms.

   

Introduction

  • The glyoxylate cycle is an anaplerotic pathway of the citric acid cycle which permits growth on two carbon compounds like acetate by bypassing the decarboxylation steps of the citric acid cycle.
  • Two enzymes play an important role in the glyoxylate cycle i.e. Isocitrate lyase and malate synthase.
  • Smith & Gunsalus (1954) reports the enzyme isocitrate lyase in the extract of Pseudomonas species.
  • Kornberg and co-workers reported the glyoxylate cycle or glyoxylate bypass.
  • The glyoxylate cycle is observed in plants, archaea, bacteria, protists, fungi and nematodes. However the presence of glyoxylate cycle in animals remains controversial.
  • It was believed that vertebrates were not capable to perform this cycle because there was no proof for the presence of its two important enzymes, isocitrate lyase and malate synthase. However, some research proposes that this cycle may present in some of the vertebrates.
  • Glyoxylate cycle allows the microorganisms to grow on two carbon compounds.
  • Glyoxylate cycle plays an important role in plants during seedlings. During the seed germination plants converts store lipid molecule in to carbohydrate using glyoxylate cycle. During seed germination photosynthesis is not operating so role of glyoxylate cycle is crucial.
  • The glyoxylate bypass allows plant seeds to stock up energy and carbon sources from fat. This stored fat is used for the generation of glucose during the germination.
  • It was believed that glyoxylate cycle operates only inside the perioxisomes of fungi and plants. However it is no longer valid. Enzymes of glyoxylate cycle are found in cytoplasm as well as in the perioxisomes.

 

Overview of Glyoxylate cycle

  • Oxaloacetate present in plant mitochondria is converted to aspartate. Aspartate then crosses the mitochondrial membrane and transport to glyoxysome.
  • Aspartate present in glyoxysome is converted back to oxaloacetate.
  • Oxaloacetate reacts with acetyl-CoA to form citrate. Citrate undergoes isomerisation to form isocitrate. (Reaction of TCA cycle)
  • Isocitrate lyase cleaves the Isocitrate to form succinate and glyoxylate.
  • Glyoxylate reacts with acetyl-CoA to form malate. This reaction is catalysed by malate synthase.
  • Malate is transported in cytoplasm. Malate is oxidized to form oxaloacetate. (Reaction of TCA cycle). This reaction is catalysed by the malate dehydrogenase.
  • Succinate produced from isocitrate is transported to mitochondria. It then enters in to TCA cycle.

 

13.1 OVERVIEW OF GLYOXYLATE CYCLE

 

 

Conversion of isocitrate to glyoxylate and Succinate by isocitrate lyase

  • The first two steps are similar to the first two reactions in the TCA cycle.
  • The glyoxylate cycle sidestep the two oxidative decarboxylation steps of the TCA cycle. It diverts the isocitrate through the isocitrate lyase and malate synthase reactions.
  • Acetyl-CoA enters the glyoxylate cycle at two steps but loss of carbon in the form of carbon dioxide is not observed.
  • In this reaction isocitrate is cleaved in to succinate and glyoxylate by the enzyme isocitrate lyase. Two steps of the TCA cycle is skipped i.e. two dehydrogenation and decarboxylation steps are bypassed which prevents the loss of two carbons. These carbons are saved in the form of glyoxylate.
  • In this reaction isocitrate is deprotonated to form succinate and glyoxylate. E.coli Isocitrate lyase contains Lys-193, Cys-195, His-197 and His-356 at catalytic active site while His-184 is plays an important role in the assembly of tetrameric enzyme.

 

13.2 Formation of succinate and glyoxylate

 

 

Conversion of Glyoxylate to malate by malate synthase

  • Malate synthase catalyse the reaction between Glyoxylate molecule and another acetyl CoA to form malate.
  • The final step in the glyoxylate cycle involves the regeneration of oxaloacetate. So one complete turn of the glyoxylate cycle forms one succinate molecule by utilizing two acetyl CoA molecules.

 

13.3 Formation of malate from glyoxylate

 

  • One of the intermediate of this cycle is glyoxylate provides the basis to name this metabolic pathway.
  • Succinate, generated using two acetyl CoA molecules, can be used to restock the TCA cycle or it can be used as precursors for amino acid biosynthesis or carbohydrate biosynthesis. So this cycle act as a link between catabolic and biosynthetic activities. It enables cells to utilise fatty acids or two carbon compounds such as ethanol or acetate as sole carbon source.

 

 

Role of Glyoxylate cycle in gluconeogenesis

  • The seed include a major store of oil in the form of triacylglycerol. It provides carbon and energy during germination of seed and growth of seedling. During germination triacylglycerol is cleaved to fatty acids by lipase. These fatty acids are then routed to specialized structure called perioxisomes. Fatty acid undergoes β-oxidation to produce acetyl-CoA. Peroxisomal acetyl-CoA is directed to gluconeogenesis through the glyoxylate cycle.
  • Glyoxylate is converted to malate by malate synthase in the glyox ysome. Oxaloacetate is formed from the oxidation of malate dehydrogenase. This oxaloacetate is utilized for the gluconeogenesis.

13.3 Glyoxylate cycle in relation to gluconeogenesis

 

 

 

Similarities with TCA cycle

  • The pathway is essentially a modified version of the citric acid cycle. The glyoxylate cycle make use of five enzymes used in the citric acid cycle. They are citrate synthase, aconitase, succinate dehydrogenase, fumarase, and malate dehydrogenase.
  • The glyoxylate cycle sidestep the two oxidative decarboxylation steps of the TCA cycle. It diverts the isocitrate through the isocitrate lyase and malate synthase reactions.
  • This bypass allows simple carbon compounds to be used as a sole source of carbon as well as the biosynthesis of macromolecules.

 

 

Why coordinate regulation is required to control glyoxylate cycle?

  • Isoenzymes of the enzymes which are common to TCA cycle and glyoxylate cycles are observed in the living system. They are specific to organelles i.e. one specific to mitochondria while the other specific to glyoxysome. Glyoxysome are not found in all plant tissues at all times. They build up in seeds during germination.
  • Three intracellular compartments carryout the transformation of dicarboxylic and tricarboxylic acids. It includes mitochondria, glyoxysome, and the cytoplasm. Metabolites are continuously interchanged in these three compartments.
  • Oxaloacetate from the mitochondria (TCA cycle) is passed to the glyoxysome in the form of aspartate. Aspartate then crosses the mitochondrial membrane and transport to glyoxysome. Aspartate present in glyoxysome is converted back to oxaloacetate. Oxaloacetate reacts with acetyl-CoA to form citrate. Citrate undergoes isomerisation to form isocitrate. (Reaction of TCA cycle). Isocitrate lyase cleaves the Isocitrate to form succinate and glyoxylate. Glyoxylate reacts with acetyl-CoA to form malate. This reaction is catalysed by malate synthase. Malate is transported in cytoplasm. Malate is oxidized to form oxaloacetate. (Reaction of TCA cycle). This reaction is catalysed by the malate dehydrogenase.
  • Succinate produced from isocitrate is transported to mitochondria. It then enters in to TCA cycle. It is transformed to malate and then oxidised to oxaloacetate. Oxaloacetate is converted to hexose via gluconeogenesis. It is then transported to growing roots and shoots.
  • It engages four different pathways. It involves fatty acid breakdown to acetyl-CoA (glyoxysome), the glyoxylate cycle (glyoxysome), the TCA cycle (mitochondria), and gluconeogenesis (cytoplasm).
  • So overall it involves different pathways and shares some common intermediates which necessitates that these pathways are regulated and coordinated together.

   

Coordinate regulation of glyoxylate cycle and TCA cycle

  • The enzymes of the glyoxylate cycle are repressed by the presence of glucose or another more rapidly utilized substrate.
  • In anaerobic condition, anaerobic respiratory control system suppresses the citric acid cycle and the glyoxylate cycle under anaerobic conditions.
  • Glyoxylate and Citric Acid Cycles shares some common intermediates which necessitate that these pathways are regulated and coordinated together. One of the crucial intermediate is Isocitrate, found at branching point amongst citric acid and glyoxylate cycles.
  • Covalent type of modification of a protein kinase phosphorylates regulates the activity of Isocitrate dehydrogenase and ultimately the dehydrogenase gets inactivated. Inactivated isocitrate dehydrogenase leads to the diversion of isocitrate into the glyoxylate cycle and from there it is diverted towards the glucose biosynthesis. The phosphate group is removed by phosphoprotein phosphatase from isocitrate dehydrogenase, which reactivates the enzyme with sending more isocitrate through the TCA cycle.
  • Lowered concentration of regulators of these cycles, results into signalling enough flux through the TCA cycle and isocitrate dehydrogenase is inactivated by the protein kinase.

   

  Table 1 List of allosteric effectors of kinase and phosphatase

 

Intermediates of the Glycolytic pathway Intermediates of the Citric acid cycle Energy depletion indicating cofactors
Phosphoenolpyruvate* Citrate AMP*
Pyruvate* Isocitrate* ADP*
3-Phosphoglycerate* α-Ketoglutarate* NADP+
Fructose-6-phosphate Oxaloacetate*

 

                        The kinase activity inhibited by all compounds shown in above table.

 

                        * stimulates the phosphatase activity

 

  • The intermediates that acts as an activator of isocitrate dehydrogenase also act as an allosteric inhibitors of isocitrate lyase
  • Under the condition where the metabolisms yield is fast and provides sufficient energy to keep the concentrations of anaplerotic molecule low, leads to the inactivation of isocitrate dehydrogenase
  • When isocitrate lyase is gets free from inhibition and isocitrate enters into the glyoxylate pathway, there it is used for synthesis of amino acids, carbohydrates and other cellular materials.

   

 

Function of the glyoxylate cycle in various organisms.

 

Plants:

  • It occurs in glyoxysome. Glyoxylate cycle plays an important role in plants during seedlings. During the seed germination plants converts store lipid molecule in to carbohydrate using glyoxylate cycle. During seed germination photosynthesis is not operating so role of glyoxylate cycle is crucial.
  • It enables plant to use acetate as a carbon and energy source.

     Fungi:

  • It plays an important role in pathogenesis of fungi. It was observed that concentration of isocitrate lyase and malate synthase is significantly increased during pathogenesis. Exact correlation is not understood.

 

 Bacteria:

  • Many bacteria have enzymes of glyoxylate and citric acid cycle in the cytoplasm. It enables them to grow on acetate as their sole source of carbon and energy.
  • Glyoxylate bypass explains the ability of bacteria, yeast, and other microorganisms to utilize acetate as a sole source of carbon for growth.
  • It plays an important role in pathogenesis of bacteria. It was observed that concentration of isocitrate lyase and malate synthase is significantly increased during pathogenesis.

 

Additional information

  • In recent studies it was observed that Isocitrate lyase, a key enzyme of glyoxylate cycle, is found to play a crucial role in persistence of Mycobacterium tuberculosis in mice. Recent studies by Sharma R. etal (2013) indicate that Isocitrate lyase can be a potential target for anti-tuberculosis drug. Experimental findings suggest that inhibitor of mycobacterium isocitrate lyase have significant antimycobacterial effect.
  • Importance of Isocitrate lyase and Malate synthase in fungal pathogens: Many scientific reports indicate that isocitrate lyase plays an important role in fungal virulence. Significant expression of Isocitrate lyase gene is observed during infection of M. grisea, the rice blast pathogen. Removal of the ICL gene results in to reduction of pathogenesis.
  • ICL plays important role in the virulence. It is further evident from study of Candida albicans.
  • Transcriptional profile of clinical specimen showed that all the genes of glyoxylate cycle is induced.
  • In Salmonella species, it is observed that ICL deficient strain is not able to cause the persistent chronic infection in mice while in case of Brucella suis glyoxylate cycle is observed to be unnecessary for virulence.
  • It is very important to generate information about this pathway and their role in pathogenesis. Such findings provide the prospect to develop specific inhibitors of isocitrate lyase and Malate synthase. This could be used to combat fungal and bacterial diseases.

 

 

you can view video on Glyoxylate cycle

 

Reference

  • H.L. Kornberg, N.B. Madsen, The metabolism of C2 compounds in microorganisms. 3. Synthesis of malate from acetate via the glyoxylate cycle, Biochem. J. 68 (1958) 549– 557.
  • M.C. Lorenz, G.R. Fink, The glyoxylate cycle is required for fungal virulence, Nature 412 (2001) 83–86.
  • Scott A. Ensign, Revisiting the glyoxylate cycle: alternate pathways formicrobial acetate assimilation, Molecular Microbiology (2006) 61(2), 274–276

 

Web site

  • http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=GLYOXYLATE-BYPASS
  • http://www.wiley.com/college/pratt/0471393878/student/weblinks/index.html (Essential biochemistry)
  • https://en.wikibooks.org/wiki/Principles_of_Biochemistry
  • http://www.unipathway.org

 

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,5th 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)
  • Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition.New York: W H Freeman; 2002.