Pyruvate Decarboxylase

There are two functions of the pyruvate decarboxylase. Pyruvate and hydroxythyl converge together while carbon dioxide molecules are eliminated. Than, hydroxyethyl group transfers and attaches to TPP to the lipoamide from the E2 component of the complex. The final transfer brings the molecule back to its original form. (Function)

“The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis” (Oba). There are 548 amino acids and very similar to that of Zymomonas Mobilis. (Raj K) “Three distinct subunit composition isoforms are apparent” (BC). There are sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Along with alpha 4, homotetrameric holoenzyme, alpha 2 beta 2, heterotetramerica holoenzyme, and beta 4 are involved. The beta 4 is sometimes in an unreported form. There are 537 CA atoms and 19 helixes in the secondary structure.

Hubner performed many experiments on the molecule and came up with this conclusion after one of his experiments.
The pH dependence of the quaternary structure of pyruvate decarboxylase (EC 4.1.1.1) has recently been discovered. In the present study we have investigated the change in quaternary structure by observing the binding of the cofactor, thiamine pyrophosphate, using 31P NMR spectroscopy. The dissociation of the native tetramers into dimers when increasing the pH coincides with a weaker binding of the cofactor and loss of enzyme activity. The results provide further evidence that thiamine pyrophosphate is bound primarily via the beta-phosphate moiety. In addition, a phosphoserine has been discovered in two of the four subunits. (Hubner)

More information was found by a scientist named Diefenbach. His experiments and examination of the molecule found a great amount of information out about it.

To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was prepared by dialysis against 1 mM-dipicolinic acid at pH 8.2. This apoenzyme was then used in a series of experiments that included determination of: (a) the affinity towards one cofactor when the other was present at saturating concentrations; (b) cofactor-binding rates by measuring the quenching of tryptophan fluorescence on the apoenzyme; (c) the effect of replacement of cofactors with various analogues; (d) the stoichiometry of bound cofactors in holoenzyme; and (e) the molecular mass of apoenzyme by gel filtration. The results of these experiments form the basis for a proposed model for the re-activation of Z. mobilis pyruvate decarboxylase apoenzyme by its cofactors. In this model there exists two alterative but equivalent pathways for cofactor binding. In each pathway the first step is an independent reversible binding of either thiamin diphosphate (Kd 187 microM) or Mg2+ (Kd 1.31 mM) to free apoenzyme. When both cofactors are present, the second cofactor-binding step to form active holoenzyme is a slow quasi-irreversible step. This second binding step is a co-operative process for both thiamin diphosphate (Kd 0.353 microM) and Mg2+ (Kd 2.47 microM). Both the apo- and the holo-enzyme have a tetrameric subunit structure, with cofactors binding in a 1:1 ratio with each subunit. (Diefenbach)

The curve for v[S], the same as pryuvate decarboxylase, shows that catalytic activity inside of the enzyme has to be regulated by a substrate. The inactive enzyme can only be activated by 2-oxo acids and 2-oxo acid amides. These cannot be a substrate inside of the enzyme. The actual dissociation constant completely depends on electrophilic nature of a carbonyl group, the structure of the activator molecules are completely independent from the saturation concentration of the catalytic activity.
The crystal structure of pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate-dependent enzyme isolated from Saccharomyces cerevisiae, has been determined and refined to a resolution of 2.3 A. Pyruvate decarboxylase is a homotetrameric enzyme which crystallizes with two subunits in an asymmetric unit. The structure has been refined by a combination of simulated annealing and restrained least squares to an R factor of 0.165 for 46,787 reflections. As in the corresponding enzyme from Saccharomyces uvarum, the homotetrameric holoenzyme assembly has approximate 222 symmetry. In addition to providing more accurate atomic parameters and certainty in the sequence assignments, the high resolution and extensive refinement resulted in the identification of several tightly bound water molecules in key structural positions. These water molecules have low temperature factors and make several hydrogen bonds with protein residues. There are six such water molecules in each cofactor binding site, and one of them is involved in coordination with the required magnesium ion. Another may be involved in the catalytic reaction mechanism. The refined model includes 1074 amino acid residues (two subunits), two thiamin diphosphate cofactors, two magnesium ions associated with cofactor binding and 440 water molecules. From the refined model we conclude that the resting state of the enzyme-cofactor complex is such that the cofactor is already deprotonated at the N4′ position of the pyrimidine ring, and is poised to accept a proton from the C2 position of the thiazolium ring (Arjunan).

The stopped-flow techniques can be looked at to evaluate which methods of activation are reversible. The controlled substrate molecule can be shown by using a glyoxylic acid as an irreversibly active-site marker.

There are 1647 base pairs contained in the gene. It has the same codon usage as manhy other glycolytic enzymes. While 100 base pairs are terminated, they usually start at -30 terminates.

Pyruvate decarboxylase is very stabile, extremely easy to purfy, has a homotetrameric subunit structure, and has a very simple kinetic property. Oxidation of pyruvate decarboxylase can happen with two routes. In pyruvate decarboxylase-negatve mutants, a pyruvate dehydrogenase complex is the only link that blycolysis and tricarboxylic acid can run.

If oxygen is present, reoxidation of NADH is not a problem. Pyruvate is decarboxylated to acetyl-CoA by the pyruvate dehydrogenase complex and the acetyl-CoA is oxidized by the Krebs cycle. Overall reaction:

pyruvate + NAD+ + CoASH � acetyl-CoA + NADH + H+ + CO2

The pyruvate dehydrogenase complex consists of three enzymes: E1 = pyruvate decarboxylase (coded by aceE gene), E2 = lipoate transacetylase (aceF gene), E3 = lipoate dehydrogenase = lipoamide dehydrogenase (lpd gene).
E1, pyruvate decarboxylase, splits pyruvate into CO2 and a 2-carbon fragment which is attached to its cofactor – thiamine pyrophosphate (TPP). The 2-carbon fragment is attached to the five membered ring and replaces the hydrogen atom which is circled in the diagram.

In certain organisms, e.g. yeast, pyruvate decarboxylase acts independently during fermentation and releases the 2-carbon fragment as acetaldehyde so converting pyruvate to acetaldehyde plus CO2. In aerobic growth, it acts as part of the PDH complex. After releasing CO2 it hands on the 2-carbon fragment (the hydroxyethyl group) to E2, the next enzyme in the pyruvate dehydrogenase complex (see diagram).

E2, lipoate transacetylase, receives the two carbon fragment. The 2-carbon fragment is attached to one of the sulfurs on the lipoic acid cofactor, and the S-S bond is opened up. Lipoic acid is covalently bound to E2 via the side-chain amino group of a lysine residue: Lipoic acid-CO-NH-lysine-E2

The two carbon fragment is then transferred to the sulfhydryl group of coenzyme A. Coenzyme A is a universal carrier of acyl groups (see diagram). CoA consists of adenosine monophosphate (adenine, ribose, phosphate) plus an extra phosphate (attached to the 3′ hydroxyl of the ribose), linked to phosphopantetheine (phosphate, pantothenic acid, cysteamine). Cysteamine is derived by decarboxylation from cysteine and provides the active sulfhydryl group.

E3, lipoamide dehydrogenase, carries an FAD cofactor which reoxidizes the lipoic acid of E2. The pyruvate dehydrogenase complex consists of a core of E2 to which the other enzymes are attached. The long lipoic acid-lysine side chain of E2 acts as a swinging arm to convey the C2 fragment from E1 to CoASH and also carries the lipoate residue past the active site of E3 for reoxidation by the FAD cofactor.

Finally, the E3-FADH2 is reoxidized to E3-FAD by transfer of its reducing equivalents to NAD: E3 – FADH2 + NAD+ âÂ?Â? NADH + H+ + E3-FAD.

In eukaryotes, pyruvate dehydrogenase is regulated by covalent modification (addition or removal of a phosphate group) – as stated in most textbooks. In bacteria this does not happen. Instead there are two other types of regulation:

a) Genetic – the PDH operon (including the aceE, aceF and lpd genes) is induced by pyruvate. The PdhR repressor protein switches the operon off when pyruvate is absent. If pyruvate is present, it binds to and inactivates the PdhR protein.
b) Enzyme activity – high levels of NADH inhibit this enzyme (NADH builds up during anaerobic conditions, especially fermentation). (Pyruvate Dehydrogenase and The Krebs Cycle)

Pyruvate decarboxylase has a few anaerobic conditions. Glycolysis producese the fates of pyruvate and NADH. Alcoholic fermentations and lactic acid is contained within and each glucose has a lot more energy. The two drawbacks are that it needs oxygen as an e-acceptor and is much slower than glycoloysis as shown in the photograph below by The Kreb Cycle:

(The Krebs Cycle)
The three stages of aerobic respiration include: a generation of Ac-CoA 2-C unit from pyruvate, an oxidation of the C’s in TCA/CAC/Krebs, and a Tfr of -through ETC & Ox-Phos

The CAC stage begins with a 2-C acetyl group. A 4-C unit is added to get a 6-C substrate. Rearrangement occurs within and thatn two of thx C’s to CO2 are oxidized. More oxidation and rearrangements occur and it goes back to the original 4-C form. The e- goes to the NAD or the FAD. A ATP is extracted and two carbons enter a cycle as acetyl and than leave directly after as CO2. The CO2 groups must be lost before most of the oxidation takes place. These stage is shown below.

(The Krebs Cycle)
The pyruvate dehydrogenase complex begins when CH2O’s enter the Krebs Cycle. The mitochondrial mix adds pyruvate and three stages take place. The equation looks similar to this:
Pyr + CoA + NAD+
To
Ac-CoA+Co2+NADH+2H+

The quaternary structure contains three types of enzymes. “There is 24 pyruvate decarboxylase a/b dimmers surrounding. There are 24 dihydrolipoyl transacetylase units and 12 dihydroplipoyl dehydrogenase in a cubical structure” (The Krebs Cycle). Five of the coenzymes are involved to thiamine pyrophosphate, Lipoic Acid, Coenzyme A and FAD.

(The Krebs Cycle)
The reaction mechanism contains different enzymes than fermentation. The enzyme removes the COO from pry -> acetaldehyde, with no net oxygen. The carboxylate leaves as carbon dioxide and other two C’s stick onto the TPP.

(The Krebs Cycle)
When it is transferred into a Lipoamide there are different things that happen. There are 5-C FA with disulfide and the ring is the oxidized form. It is reduced form and has the disulfide bond split. The acid in amide link with e-amino group of a K.

Enzymatic reaction of: decarboxylase
indolepyruvate <=> CO2 + indole acetaldehyde
The reaction direction shown, that is, A + B <==> C + D versus C + D <==> A + B, is in accordance with the direction of the reaction within a pathway.
In Pathways: tryptophan degradation

Enzymatic reaction of: decarboxylase
phenylpyruvate <=> phenylacetaldehyde + CO2
The reaction direction shown, that is, A + B <==> C + D versus C + D <==> A + B, is in accordance with the direction of the reaction within a pathway.
In Pathways: phenylalanine degradation

Enzymatic reaction of: decarboxylase
2-keto-3-methyl-valerate <=> amyl alcohol + CO2
The reaction direction shown, that is, A + B <==> C + D versus C + D <==> A + B, is in accordance with the direction of the reaction within a pathway.
In Pathways: isoleucine degradation

Enzymatic reaction of: pyruvate decarboxylase
a 2-oxo acid + pyruvate <=> CO2 + acetaldehyde
The reaction direction shown, that is, A + B <==> C + D versus C + D <==> A + B, is in accordance with the direction of the reaction within a pathway.
In Pathways: glucose fermentation

Works Cited

Arjunan, P. “Crystal structure of the thiamin diphosphate-dependent enzyme pryuvate decarboxylase from the yeast Saccharomyces cerevisiae at 3.2 A resolution.” National Library of Medicine 256 (1996). 15 Nov 2005 .

BC, Farrenkpof. “Resolution of brewer’s yeast pyruvate decarboxylase into ulitple isoforms with similar subunit structure and activity using igh-performance liquid chromatography.” National Library of Medicine 3 Apr 1992. 13 Nov 2005 .

Diefenbach, RJ. “Pryuvate Decarboxylase from zymomonas mobilis. Structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion.” Biochemical Journal 276 (1992). 13 Nov 2005 .

Function and Reaction Mechanism. Chem.uwec.edu. 13 Nov. 2005 .

Hubner, G. “Correlation of cofactor binding and the quaternary structure of pyruvate decarboxylase as revealed by 31P NMR spectroscopy.” National Library of Medicine 7 DEC 1992. 13 Nov 2005 .

Oba, K. “Purification and Characterization of Pyruvate Decarboxylase from Sweet Potato Roots.” Journal of Biochemistry 77 (1975). 13 Nov 2005 .

“Pyruvate Dehydrogenase and The Krebs Cycle.” Pyruvate Dehydrogenase and The Krebs Cycle. Science.siu.edu. 15 Nov. 2005 .

Raj K, Chandra. “Pyruvate Decarboxylase: A Key Enzyme for the Oxidative Metabolism of Lactic Acid by Acetobacter Pasteurianus.” National Library of Medicine 25 Sep 2001. 13 Nov 2005 .

“The Krebs Cycle.” The Krebs Cycle. bmb.psu.edu. 13 Nov. 2005 .

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