Pyruvate decarboxylase
Encyclopedia
Not to be confused with pyruvate dehydrogenase
, the enzyme which catalyses the link reaction.
Pyruvate decarboxylase is a homotetrameric enzyme
that catalyses
the decarboxylation of pyruvic acid
to acetaldehyde
and carbon dioxide
in the cytoplasm. It is also called 2-oxo-acid carboxylase, alpha-ketoacid carboxylase, and pyruvic decarboxylase. In anaerobic conditions, this enzyme is part of the fermentation process that occurs in yeast, especially of the Saccharomyces
genus, to produce ethanol
by fermentation. Pyruvate decarboxylase starts this process by converting pyruvate into acetaldehyde and carbon dioxide. Pyruvate decarboxylase depends on cofactors
thiamine pyrophosphate
(TPP) and magnesium. This enzyme should not be mistaken for the unrelated enzyme pyruvate dehydrogenase
, an oxidoreductase , that catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA
.
This enzyme is a homotetramer, and therefore has four 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).
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.
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 ylide
. 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.
Also included are Asp-444 and Asp-28 which 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 Ångstroms 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
. 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.
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 Cys-152, Asp-28, His-114, His-115, and Gln-477.
The normal catalytic rate of pyruvate decarboxylase is kcat = 10 s−1. However, the rate of the enzyme with a Glu-51 mutation to Gln is 1.7 s−1.
TPP has an acidic 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. The TPP can then act as a nucleophile with the loss of this C2 hydrogen, forming the ylide form of TPP. This ylide 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. Specifically, the pyridyl nitrogen N1' and the 4'-amino group of TPP are essential for the catalytic function of the enzyme-TDP complex.
Pyruvate dehydrogenase
Pyruvate dehydrogenase complex is a complex of three enzymes that transform pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. Acetyl-CoA may then be used in the citric acid cycle to carry out cellular respiration, and this complex links the glycolysis metabolic pathway to the...
, the enzyme which catalyses the link reaction.
Pyruvate decarboxylase is a homotetrameric enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that catalyses
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
the decarboxylation of pyruvic acid
Pyruvic acid
Pyruvic acid is an organic acid, a ketone, as well as the simplest of the alpha-keto acids. The carboxylate ion of pyruvic acid, CH3COCOO−, is known as pyruvate, and is a key intersection in several metabolic pathways....
to acetaldehyde
Acetaldehyde
Acetaldehyde is an organic chemical compound with the formula CH3CHO or MeCHO. It is one of the most important aldehydes, occurring widely in nature and being produced on a large scale industrially. Acetaldehyde occurs naturally in coffee, bread, and ripe fruit, and is produced by plants as part...
and carbon dioxide
Carbon dioxide
Carbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
in the cytoplasm. It is also called 2-oxo-acid carboxylase, alpha-ketoacid carboxylase, and pyruvic decarboxylase. In anaerobic conditions, this enzyme is part of the fermentation process that occurs in yeast, especially of the Saccharomyces
Saccharomyces
Saccharomyces is a genus in the kingdom of fungi that includes many species of yeast. Saccharomyces is from Greek σάκχαρ and μύκης and means sugar fungus. Many members of this genus are considered very important in food production. One example is Saccharomyces cerevisiae, which is used in making...
genus, to produce ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
by fermentation. Pyruvate decarboxylase starts this process by converting pyruvate into acetaldehyde and carbon dioxide. Pyruvate decarboxylase depends on cofactors
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....
thiamine pyrophosphate
Thiamine pyrophosphate
Thiamine pyrophosphate , or thiamine diphosphate , is a thiamine derivative which is produced by the enzyme thiamine pyrophosphatase. Thiamine pyrophosphate is a coenzyme that is present in all living systems, in which it catalyzes several biochemical reactions...
(TPP) and magnesium. This enzyme should not be mistaken for the unrelated enzyme pyruvate dehydrogenase
Pyruvate dehydrogenase
Pyruvate dehydrogenase complex is a complex of three enzymes that transform pyruvate into acetyl-CoA by a process called pyruvate decarboxylation. Acetyl-CoA may then be used in the citric acid cycle to carry out cellular respiration, and this complex links the glycolysis metabolic pathway to the...
, an oxidoreductase , that catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA
Acetyl-CoA
Acetyl coenzyme A or acetyl-CoA is an important molecule in metabolism, used in many biochemical reactions. Its main function is to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production. In chemical structure, acetyl-CoA is the thioester...
.
Yeast
In yeast, pyruvate decarboxylase acts independently during anaerobic fermentation and releases the 2-carbon fragment as acetaldehyde plus carbon dioxide. Pyruvate decarboxylase creates the means of CO2 elimination, which the cell dispels. The enzyme is also means to create ethanol, which is used as an antibiotic to eliminate competing organisms. 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.Structure
Pyruvate decarboxylase occurs as a dimer of dimers with two active sites shared between the monomers of each dimer. The enzyme contains a beta-alpha-beta structure, yielding parallel beta-sheets. It contains 563 residue subunits in each dimer; the enzyme has strong intermonomer attractions, but the dimers loosely interact to form a loose tetramer.Active site residues
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.
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 ylide
Ylide
An ylide or ylid is a neutral dipolar molecule containing a formally negatively charged atom directly attached to a hetero atom with a formal positive charge , and in which both atoms have full octets of electrons. Ylides are thus 1,2-dipolar compounds...
. 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.
Also included are Asp-444 and Asp-28 which 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 Ångstroms away from each site) and His-92 trigger a conformational change
Conformational change
A macromolecule is usually flexible and dynamic. It can change its shape in response to changes in its environment or other factors; each possible shape is called a conformation, and a transition between them is called 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
Regulatory site
A regulatory site is a site on an allosteric protein to which a modulator molecule binds. A ligand-binding site on a receptor or enzyme distinct from the active site. Allosteric modulators alter enzyme activity by binding to the regulatory site. Also known as an "allosteric site"....
. 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.
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 Cys-152, Asp-28, His-114, His-115, and Gln-477.
The normal catalytic rate of pyruvate decarboxylase is kcat = 10 s−1. However, the rate of the enzyme with a Glu-51 mutation to Gln is 1.7 s−1.
TPP prosthetic group
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.TPP has an acidic 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. The TPP can then act as a nucleophile with the loss of this C2 hydrogen, forming the ylide form of TPP. This ylide 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. Specifically, the pyridyl nitrogen N1' and the 4'-amino group of TPP are essential for the catalytic function of the enzyme-TDP complex.