Pyruvate decarboxylase

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Pyruvate decarboxylase is a homotetrameric enzyme (EC 4.1.1.1) that catalyses the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. It is also called 2-oxo-acid carboxylase, alpha-ketoacid carboxylase, and pyruvic decarboxylase [1]. In aerobic conditions, this enzyme is the first in a three enzyme complex known as the pyruvate dehydrogenase complex, which converts pyruvate (the product of glycolysis) into acetyl CoA. In anaerobic conditions, this enzyme is part of the fermentation process that occurs in yeast to produce alcohol (this enzyme is not in animals). Pyruvate decarboxylase starts this process by converting pyruvate into acetaldehyde and carbon dioxide[2]. To do this, two thiamine pyrophosphate (TPP) and two Magnesium ions are required as a cofactors. Image:1pvd with TPP.jpg

Pyruvate decarboxylase with TPP attached

Contents

[edit] Structure

This enzyme contains a beta-alpha-beta structure, yielding parallel beta-sheets. It contains 563 residue subunites in each dimer; the enzyme has strong intermonomer attractions, but the dimers loosely interact to form a loose tetramer.[3]

3D structure image

3D image of enzyme

[edit] Active Site

the active site of pyruvate decarboxylase with selected amino acids: Glu 51, Glu 477, Asp 444, and Asp 28. Also included is cofactors TPP and Mg2+

Active site of pyruvate decarboxylase with selected amino acid residues: Glu 51, Glu 477, Asp 444, Asp 28. Also included is cofactors TPP and Mg2+.

[edit] Residues in Active Site

This enzyme is a dimer, and therefore has two active sites. The active sites are inside a cavity in the core of the enzyme where hydrogen bonding can occur and where the pyruvate reacts with TPP. Each active site has 20 amino acids, including the acidic Glu 477 (contributes to the stability of the TPP ring) and Glu 51 (aids in cofactor binding). These Glutamates also contribute to forming the TPP ylid, acting as proton donators to the TPP aminopyrimidine ring. The microenvironment around this Glu 477 is very nonpolar, contributing to a higher than normal pKa (normal Glu and Asp pKa's are around 4.6 in small proteins).[4] The lipophilic residues Ile 476, Ile 480 and Pro 26 contribute to the nonpolarity of the area around Glu 477. The only other negatively charged residue apart from TPP coenzyme is the Asp 28, which also aids in increasing the pKa of Glu 477. Thus, the environment of the enzyme must allow for the protonation of the gamma-carboxyl group of Glu 477 to be around pH 6.[5].

The aminopyrimidine ring on TPP acts as a base, once in its imine form, to pull off the C2 proton from TPP to form the nucleophile ylid.[6] This must occur because the enzyme has no basic side chains present to deprotonate the TPP C2. A mutation at the active site involving these Glu can result in the inefficiency or inactivity of the enzyme. This inactivity has been proven in experiments in which either the N1' and/or 4'-amino groups are missing. In NMR analysis, it has been determined that when TPP is bound to the enzyme along with the substate-analog pyruvamide, the rate of ylid formation is greater than the normal enzyme rate. Also, the rate of mutation of Glu 51 to Gln reduces this rate significantly.[7].

[edit] Residues in Conformational Site

Also included are Asp 444 and Asp 28- these stabilize the active site. These act as stabilizers for the Mg2+ ion that is present in each active site. To ensure that only pyruvate binds, two Cys 221 (more than 20 Angstroms away from each site) and His 92 trigger a conformational change which inhibits or activates the enzyme depending on the substrate that interacts with it. If the substrate bound in the active site is pyruvate, then the enzyme is activated by a conformational change in this regulatory site[8]. The conformational change involves a 1,2 nucleophilic addition. This reaction, the formation of a thioketal, transforms the enzyme from its inactive to active state.

Image:Ja951830tf00015.gif

Addition nucleophile Cys to change conformation of enzyme for activation

Inhibition of the site is done by a XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds as cinnamaldehydes. Other potential nucleophilic sites for the inhibitor include Cyst152, D28, His114, His115, and Gln477.[9]

Image:Hist and cyst edited.jpg

positions of His and Cys residues in respect to active site (TPP and Mg) that participate in conformation changes when interacting with pyruvate substrate.

[edit] TPP

The cofactor TPP, C12 H18 N4 O7 P2 S, is needed for this reaction's mechanism; it acts as the prosthetic group to the enzyme. The carbon atom between the sulfur and nitrogen atoms on thiazole ring act as carbanion which binds to the pyruvate. [10] TPP has an acidix H+ on its C2 that acts as the functional part of the thiazolium ring; the ring acts as an "electron sink", enabling the carbanion electrons to be stabilized by resonance[11]. The TPP can then act as a nucleophile with the loss of this C2 hydrogen, forming the ylid form of TPP. This ylid can then attack pyruvate, which is held by the enzyme pyruvate decarboxylase. During the decarboxylation of pyruvate, the TPP stabilizes the carbanion intermediates as an electrophile by noncovalent bonds[12]. Specifically, the pyridyl nitrogen N1' and the 4'-amino group of TPP are essential for the catalytic function of the enzyme-TDP complex.[13]

[edit] Function Human

In aerobic conditions, this "pre-Citric Acid Cycle" enzyme has the task of converting pyruvate into acetaldehyde and hydroxethyl-TPP to funnel into the Citric Acid Cycle.

[edit] Function in Yeast

In anaerobic conditions, the enzyme has a twofold task: first to convert pyruvate into hydroxyethyl-TPP and next to transfer the hydroxyethyl group attached to TPP to the lipoamide from the E2 component of the pyruvate dehydrogenase complex. This transfer ultimately brings pyruvate decarboxylase back to its native form, ready to catalyze the next reaction. This reaction takes place in the mitochondrial matrix.[14] In yeast, pyruvate decarboxylase acts independently during fermentatino and releases the 2-carbon fragment as acetaldehyde plus carbon dioxide. Pyruvate decarboxylase creates the means of CO2elimination, which the cell dispels. [15]. The enzyme is also means to create ethanol, which is used as an antibiotic to eliminate competing organisms[16]. The enzyme is necessary to help the decarboxylation of alpha-keto acids because there is a build-up of negative charge that occurs on the carbonyl carbon atom in the transition state; therefore, the enzyme provides the suitable environment for TPP and the alpha-keto acid (pyruvate) to meet[17]

[edit] Mechanism

The enzyme splits pyruvate into carbon dioxide and a 2-carbon fragment which is attached to its cofactor TPP. This 2-carbon fragment is attached to the five membered TPP ring in its ylid form.

pyruvate + thiamine pyrophasphate (TPP) --> hydroxyethyl-TPP + CO2 Image:Pyruvate decarb 1.jpg[18]

[edit] Overall mechanism

In aerobic conditions, the next step involves the process as follows: hydroethyl-TPP + lipoamide --> TPP + acetyllipoamide. The hydroxyethyl-TPP is oxidized by the disulfide group of the lipoamide from dihydrolipoyl transacetylase. The pyruvate decarboxylase then transfers the hydroxyethyl-TPP to the lipoamide, which brings the pyruvate decarboxylase enzyme back to its original form to catalyze the next reaction. The acetaldehyde is then converted into ethanol by the enzyme alcohol dehydrogenase and NADH. [19].

[edit] Rate of Catalytic Effect

The normal catalytic rate of pyruvate decarboxylase is kcat = 10s-1. However, the rate of the enzyme with a Glu 51 mutation to Gln is 1.7s-1.[20]

[edit] References

  1. ^ "NiceZyme View of ENZYME: EC 4.1.1.1." ExPASy Proteomics Server. Online 3 Dec 2007. http://us.expasy.org/enzyme/4.1.1.1
  2. ^ McMurry, John.The Organic Chemistry of Biological Pathways. p 179
  3. ^ Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)
  4. ^ Lobell, Mario and David H.G. Crout Pyruvate decarboxylase: a molecular modeling study of pyruvate decarboxylation and acyloin formation Journal of American Chemistry Society 1996: 118(8) 1867-1873
  5. ^ Lobell, Mario and David H.G. Crout Pyruvate decarboxylase: a molecular modeling study of pyruvate decarboxylation and acyloin formation Journal of American Chemistry Society 1996: 118(8) 1867-1873
  6. ^ Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)}}
  7. ^ Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)}}
  8. ^ Baburin I, Dikdan G, Guo F, Tous GI, Root B, Jordan F Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions Biochemistry 1998 Feb 3: 37(5)1245-1255
  9. ^ Baburin I, Dikdan G, Guo F, Tous GI, Root B, Jordan F Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions Biochemistry 1998 Feb 3: 37(5)1245-1255
  10. ^ http://chemistry.gsu.edu/glactone/vitamins/b1/tpp.gif
  11. ^ McMurry, John.The Organic Chemistry of Biological Pathways. p 179
  12. ^ Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)}}
  13. ^ Lobell, Mario and David H.G. Crout Pyruvate decarboxylase: a molecular modeling study of pyruvate decarboxylation and acyloin formation Journal of American Chemistry Society:1996. p.1867-1873
  14. ^ Stryer, Lubert Biochemistry Fourth Edition:1992p.514-518
  15. ^ Hoberg, John. "Pyruvate Decarboxylase". University of Wisconsin-Eau Claire: Chem 406. Online Dec 3 2007. http://www.chem.uwec.edu/Webpapers_F98/hoberg/hoberg.html
  16. ^ Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24
  17. ^ Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24
  18. ^ http://www.biochem.arizona.edu/.jpg
  19. ^ Hoberg, John. "Pyruvate Decarboxylase". University of Wisconsin-Eau Claire: Chem 406. Online 3 Dec 2007. http://www.chem.uwec.edu/Webpapers_F98/hoberg/hoberg.html
  20. ^ Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)

[1] "NiceZyme View of ENZYME: EC 4.1.1.1." ExPASy Proteomics Server. Online 3 Dec 2007. http://us.expasy.org/enzyme/4.1.1.1

[2] Clark, David."Pyruvate dehydrogenase and the Krebs Cycle". SIUC Dept. Microbiology. Online 3 Dec 2007. http://www.science.siu.edu/microbiology/micr425.

[3] McMurry, John.The Organic Chemistry of Biological Pathways. p 179

[4] Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)

[5] Lobell, Mario and David H.G. Crout Pyruvate decarboxylase: a molecular modeling study of pyruvate decarboxylation and acyloin formation Journal of American Chemistry Society 1996: 118(8) 1867-1873

[6] Lobell, Mario and David H.G. Crout Pyruvate decarboxylase: a molecular modeling study of pyruvate decarboxylation and acyloin formation Journal of American Chemistry Society 1996: 118(8) 1867-1873

[7] Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)

[8] Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)

[9] Baburin I, Dikdan G, Guo F, Tous GI, Root B, Jordan F Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions Biochemistry 1998 Feb 3: 37(5)1245-1255

[10] Baburin I, Dikdan G, Guo F, Tous GI, Root B, Jordan F Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions Biochemistry 1998 Feb 3: 37(5)1245-1255

[11] http://chemistry.gsu.edu/glactone/vitamins/b1/tpp.gif

[12] McMurry, John.The Organic Chemistry of Biological Pathways. p 179

[13] Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)

[14] Lobell, Mario and David H.G. Crout Pyruvate decarboxylase: a molecular modeling study of pyruvate decarboxylation and acyloin formation Journal of American Chemistry Society:1996. p.1867-1873

[15] Hoberg, John. "Pyruvate Decarboxylase". University of Wisconsin-Eau Claire: Chem 406. Online Dec 3 2007. http://www.chem.uwec.edu/Webpapers_F98/hoberg/hoberg.html

[16] Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)

[17] Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)

[18] http://www.biochem.arizona.edu/

[19] Hoberg, John. "Pyruvate Decarboxylase". University of Wisconsin-Eau Claire: Chem 406. Online 3 Dec 2007. http://www.chem.uwec.edu/Webpapers_F98/hoberg/hoberg.html

[20] Dyda, F : Furey, W : Swaminathan, S : Sax, M : Farrenkopf, B : Jordan, Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution.Biochemistry. 1993 Jun 22; 32(24)

[edit] External links

v • d • e
Carbon-carbon lyases (EC 4.1)
4.1.1: Carboxy-lyases
4.1.2: Aldehyde-lyases
4.1.3: Oxo-acid-lyases