9 Conversion of pyruvate to Acetyl CoA

Dr. Ramesh Kothari

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Conversion Of Pyruvate To Acetyl Co – A

 

OBJECTIVES

  1. To understand the entry of pyruvate into TCA cycle
  2. To examine the metabolic fate of pyruvate
  3. To understand importance of PDH

   

INTRODUCTION

  • Pyruvate is the common product of sugar catabolisms in all the major carbohydrate catabolic pathways.
  • One of the fates of pyruvate is to be oxidized to acetyl-CoA.
  • Pyruvate is is catalyzed in anaerobically growing bacteria by either pyruvate ferredoxin oxidoreductase or pyruvate formate lyase .
  • An acetyl CoA thus formed is change in to acetyl phosphate by enzyme phosphotransacetylase and the acetyl phosphate donates the phosphoryl group to ADP in a substrate leval phosphorylation catalyzed by acetate kinase.
  • The acetate thus formed is excreted.
  • However ,certain anaerobic archaea oxidized pyruvate to acetyl CoA by using pyruvate ferredoxin oxidoreductase and then oxidized the acetyl -CoA to CO2 via citric acid cycle.

 

PYRUVATE DEHYDROGENASE

  • The oxidation of pyruvate to acetyl -CoA during aerobic growth is carried out by the enzyme complex pyruvate dehydrogenase , which is widespread in both prokaryotes and eukaryotes
  • The acetyl – CoA formed during aerobic growth is oxidized to CO2 in the citric acid cycle .
  • The overall reaction for pyruvate dehydrogenase is :
  • The pyruvate dehydrogenase complex is a very large enzyme complex (in E.coli, about 1.7 times the size of the ribosome) located in the mitochondria of eukaryotic cell and in the cytosol of prokaryotes .
  • Pyruvate dehydrogenase complex is similar to the α ketoglutarate dehydrogenase complex which is useful enzyme in citric acid cycle and α ketoglutarate dehydrogenase complex is also important for the metabolisms of some aminoacid like leucine, valine and isoleucine.
  • The pyruvate dehydrogenase complex containing 3 enzyme E1,E2 and E3. The E1 and E2 genes are specific for the different types of substrates, while E3 gene is used for each of enzyme complexes.
  • 60 E2 polypeptides present in eukaryotes and gram-positive bacteria,which form a symmetrical icosahedral complex. The E1 (αβ)2 tetramers and E3 dimers then associate with the E2 complex. In mammals there are other 3 protein are there inside the complex.
  • These three protein are protein X, E1-kinase, and phospho-E1-phosphatase .
  • E1-kinase, and phospho-E1-phosphatase are the regulatory protein where as protein X is necessary for binding of E3 enzyme to E2 complex.
  • The pyruvate dehydrogenase from E.coli consist of 24 molecules of enzyme E1( pyruvate dehydrogenase), 24 molecules of enzyme E2 (dihydrolipoate transacetylase), and 12 molecules of enzyme E3 (dihydrolipoate dehydrogenase ).
  • For each type of the α-ketoacid dehydrogenase, the enzyme -1is needed for the specific correlation and enzyme-2 to permit activity.
  • The E1 are not able to donate acetyl groups to free lipoic acid. That’s whyenzyme E1 came in to view to identify both the lipoyl-lysine and the lipoyl-lysine attached protein.
  • Loss of the acetyl group is prevent by this, and also play a role in regulation of the complex. Eukaryotes having one and E.coli having three,80 residue of lipoyl domain present in each E2 polypeptide. That are joint to the core of the complex by flexible domains within the E2 peptide.
  • The combination of the supple E2 and the lipoyl-lysine group (which in itself is 14Å long), allow each E2 prosthetic group to react with the complex active site. E2 having a flexible domain at the core of complex where that attech.
  • The E1 binds pyruvate, and then forms a covalent bond between the TPP cofactor and the two carbon hydroxyethyl group remaining after decarboxylation of the pyruvate.
  • (Note: the name of E1 and complex are similar that is pyruvate dehydrogenase but E1 is not a dehyrogenase so to avoid confusion pyruvate decarboxylase is used for this enzyme )
  • Several very important cofactor are involved.
  • The cofactor are thiamine pyrophosphate (TPP),derive from the vitamin thiamin.
  • Flavin adenine dinucleotide (FAD) ,derive from vitamin riboflavin .
  • Lipoic acid (RS2); nicotinamide adenine dinucleotide (NAD +), derived from the vitamin nicotinamide .
  • Coenzyme A, derive from the vitamin pantothenic acid .
  • The large size of the complex is presumably designed to process the heavy stream of pyruvate that is generated during the catabolisms of sugar and other compound.

 

REACTIONS OF PDH COMPLEX

  • The payruvate dehydrogenase complex catalyzes a short metabolic pathway rather then simply a single reaction.
  • The individual reaction carried out by pyruvate dehydrogenase complex are as follow

 

STEP-1

  • Pyruvate is decarboxylated to form active acetaldehyde bound to TPP.
  • The reaction is catalysed by pyruvate dehydrogenase (E1)
  • The E1 attach with pyruvate, and then forms a covalent bond between the thiamin pyrophosphate cofactor and the two carbon hydroxyethyl group remaining after decarboxylation of the pyruvate.
  • The name of E1 and complex are similar that is pyruvate dehydrogenase but E1 is not a dehyrogenase so to avoid confusion pyruvate decarboxylase is used for this enzyme that forms acetaldehyde from pyruvate in microorganisms, the term pyruvate decarboxylase is also used in some literature for the E1 enzyme of the pyruvate dehydrogenase complex
  • The enzyme E1 has a very low affinity for the carbon dioxide, because of this reversal of decaroxylation is very improbable ;carbon dioxide is release, because of this E1 reaction is irreversible and therefore the entire pyruvate dehydrogenase is irreversible .

 

STEP-2

  • The active acetaldehyde is oxidized to the level of carboxyl by the disulfide in lipoic acid.
  • The disulfide of lipoic acid is reduced to sulfhydral.
  • During the reaction, TPP is displaced and the acetyl group is transferred to the lipoic acid.
  • The reaction is also catalyzed by pyruvate dehydrogenase.
  • The hydroxyl group from thiamin pyrophosphate cofactor of E1 transfer to lipoamide which is prosthetic group of E2 by second enzyme dihydrolipoyl transacetylase or dihydrolipoyl acetyltransferase (E2). Transfer of hydroxyl group form the acetyle lipoamide bound to the enzyme.
  • Acetyl group is transfersed by Enzyme 2 to Coenzyme A, which is released as acetyl-CoA.
  • (Note: The acetyl group and the CoA form bond which need a high energy; in this case to make bond energy is come from pyruvate . In contrast, acetyl-CoA formation from acetate requires the use of ATP.)

  STEP-3

  • A trans acetylation occur in which lipoic acid is displaced by CoASH, forming acetyl – CoA and reduced lipoic acid .
  • The reaction is catlysed by dihydrolipoate transacetylase E2. lipoamide (dihydrolipoamide) is reduced as a result of the acetyl-CoA formation .
  • The dihydrolipoamide oxidizes to form oxidized lipoamide by the the third enzyme, dihydrolipoyl dehydrogenase (E3), the starting form of the E2.

    STEP-4

  • The lipoic acid is oxidized by dihydrolipoate dehydrogenase, E3- FAD.

 

 

STEP-5

  • The E3 – FAD transfers the electrons to NAD+.

 

The mechanisms of action of pyruvate dehydrogenase complex .

  • All the intermediates remain bounds to the complex and are passed from one active site to another. Presumably, this offers the advantage inherent in all multienzyme complex.
  • In mammals, the PDH complex has an approximate molecular weight of 9 x 106 N. lt contains 60 molecules of dihydrolipoyl transacetylase and about 20-30 molecules each of the other two enzymes (pyruvate dehydrogenase and dihydrolipoyl dehydrogenase).
  • A comparable enzyme with PDH is a α-ketoglutarate dehydrogenase complex of citric acid cycle which catalyses the oxidative decarboxylation of a-ketoglutarate to succinyl CoA.
  • Arsenic poisoning : The enzymes PDH and a-ketoglutarated dehydrogenasea are inhibited by arsenite.
  • Arsenite binds to thiol (-SH) groups of lipoic acid and makes it unavailable to serve as cofactor.

 

REGULATION OF PDH

  • Pyruvate is a more important than acetyl CoA, so to prevent unrequired cleavage of pyruvate, regulation of irreversible pyruvate dehydrogenase reaction is necessary.
  • In pyruvate dehydrogenase complx, E1 reaction is irreversible, this mean that E1reaction is important for prevent of un necessary cleavage of pyruvate .
  • Pyruvate dehyrogenase complex is inhibited by acetyl CoA and acetyl CoA is the product of E1 reaction which is produced by cleavage of fatty acid.
  • Pyruvatedehydrogenasies a good example of end product (acetyl CoA, NADH) inhibition’ Besides this, PDH is also regulated by phosphoryIa tion and dephosphoryaltion .
  • PDH is active as a dephosphoenzyme while it is inactive as a phosphoenzyme PDH phosphatase activity is promoted by Ca+2, Mg+ and insulin (in adipose tissue). lt is of interest to note that calcium released during muscle contraction stimulates PDH (by increasing phosphatase activity) for energy Production.

 

Regulation of pyruvate dehdrogenase complex

 

  • PDH kinase (responsible to form inactive PDH) is actively encourage by ATP, NADH and acetyl CoA, while it is hinder by NAD+, CoA and pyruvate.The net result is that in the presence of high energy signals (ATP, NADH), the PDH is offened.

 

BIOCHEMICAL IMPORTANCE OF PDH

  1. Lack of TPP (due to deficiency of thiamine) inhibits PDH activity resulting in the accumulation of Pyruvate.
  2. ln the thiamine lacking alcoholics, pyruvate is rapidly turn in to lactate, resulting in lactic acidosis.
  3. ln patients with inherited deficiency of PDH, lactic acidosis (usually after glucose load) is observed.
  4. PDH activitvy can be inhibited by arsenic and mercuric ions. This is brought about by binding of these ions with -SH groups of lipoic acid.

 

 

METABOLIC IMPORTANCE OF PYRUVATE

  • Pyruvate is a key metabolite. Besides its conversion to acetyl CoA (utilized in a wide range of metabolic reactions-citric acid cycle, fatty acid synthesis etc.), pyruvate is a good substratef or gluconeogenesis.

 

you can view video on Conversion of pyruvate to Acetyl CoA

 

 

References

Research papers

 

  • Ciszak EM, Korotchkina LG, Dominiak PM, Sidhu S, Patel MS (June 2003). “Structural basis for flip-flop action of thiamin pyrophosphate-dependent enzymes revealed by human pyruvate dehydrogenase”. J. Biol. Chem. 278 (23): 21240–6
  • Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco; Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (October 2004). “UCSF Chimera—a visualization system for exploratory research and analysis”. J Comput Chem 25 (13): 1605–12
  • Jaimes, R 3rd (Jul 2015). “Functional response of the isolated, perfused normoxic heart to pyruvate dehydrogenase activation by dichloroacetate and pyruvate.”. Pflugers Arch.
  • Arjunan P, Nemeria N, Brunskill A, Chandrasekhar K, Sax M, Yan Y, Jordan F, Guest JR, Furey W. (2002). “Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution”. Biochemistry 41 (16): 5213– 21
  • Leung PS, Rossaro L, Davis PA, et al. (2007). “Antimitocho -ndrial antibodies in acute liver failure: Implications for primary biliary cirrhosis”. Hepatology 46 (5): 1436–42
  • Recny MA, Hager LP (1982). “Reconstitution of native Escherichia coli pyruvate oxidase from apoenzyme monomers and FAD”. J. Biol. Chem. 257 (21): 12878–86

 

Web

  • www.rose-hulman.edu