Pyruvate decarboxylase
Pyruvate decarboxylase is a homotetrameric enzyme (EC 4.1.1.1 ) that catalyzes the decarboxylation of pyruvic acid to form acetaldehyde and carbon dioxide in the cytoplasm. Under anaerobic conditions, this enzyme is part of the fermentation that occurs in yeasts, especially in the genus Saccharomyces to produce ethanol by fermentation. The conversion of pyruvate to acetaldehyde and carbon dioxide by pyruvate decarboxylase is at the beginning of this process. Pyruvate decarboxylase is dependent on the cofactor thiamin pyrophosphate, and magnesium. The enzyme should not be confused with the pyruvate dehydrogenase, an oxidoreductase ( EC 1.2.4.1 ), which catalyzes the oxidative decarboxylation of pyruvate to acetyl -CoA.
Yeast
In yeast pyruvate decarboxylase operates independently during the anaerobic digestion and gives as the C2 body from acetaldehyde and carbon dioxide. Pyruvate decarboxylase represents a way to reduce CO2 elimination, the donor cell. The ethanol produced by the enzyme used as an antibiotic, which is used to eliminate competing organisms the enzyme is needed to assist in the decarboxylation of alpha-keto acids, as accumulated in the transient state at the carbonyl carbon atom increases the negative charge; the enzyme thus provides the optimal environment so that thiamine and alpha-keto acids ( pyruvate ) to take.
Structure
Pyruvate decarboxylase is constructed as a dimer of dimers with two active sites, each formed from two monomers each dimer. The enzyme contains a beta- alpha-beta structure, which leads to parallel beta-sheets and 563 residue in each subunit dimer. The monomers are joined together by strong interactions to form dimers, dimers interact but only loosely, in order to form a loose tetramer.
The active site
Pyruvate decarboxylase is a homotetramer and thus has four active centers. The active sites are located in a cavity in the core region of the enzyme, where hydrogen bonds can be formed and where the pyruvate with thiamin pyrophosphate (TPP ) reacts. Each active site is composed of 20 amino acids, including Glu -477 ( contributes to the stability of the TPP ring in ) and Glu -51 ( helps in the cofactor - binding). This Glutamate also help to make the TPP ylide, by acting as Protondonator against the TPP Aminopyrimidinring. The microenvironment around Glu 477 is very nonpolar, by a higher pKa than usual ( normally the pK of Glu and Asp in small proteins about 4.6 ).
The lipophilic residues Ile -476, Ile -480 and Pro -26 contribute to the non-polarity of the region around Glu -477. The only other negatively charged moiety, apart from TPP coenzyme, Asp -28, which also helps to increase the pKa of Glu -477. So must allow at a pH around 6, the environment of the enzyme protonation of the gamma -carboxyl group of Glu -477.
Of the TPP Aminopyrimidinring deprotonated when it is present as the imine, the C2 atom of TPP, so as to form the nucleophilic ylide. This must be done in this way, since the enzyme does not have basic side chains, in order to deprotonate the C2 of TPP. A mutation in the active site in connection with Glu can lead to inefficiency or inactivity of the enzyme. This lack of action has been demonstrated in experiments in which either the N1 ' and / or 4'- amino group were missing. In NMR analysis, it was found that when TPP is attached to the enzyme to the substrate together with the analog Pyruvamide, the rate of ylide formation is greater than that of the normal enzyme. In addition, 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. They act as stabilizers for the Mg2 ion, which is located in each active site. To ensure that only pyruvate binds to solve two Cys -221 (more than 20 Å away from each center ) and His- 92 from a conformational change that inhibits or activates the enzyme, depending on the substrate interacting with him. When the substrate is bound in the active site of pyruvate, then the enzyme will be activated by a conformational change of the regulatory site. The conformational change involves a 1,2 nucleophilic addition. This reaction, the formation of a thioketal, converts the enzyme from an inactive to an active form.
The inhibition of the site is initiated by an inhibitor or a substrate analogue of the mold XC6H4CH = CHCOCOOH, as well as by products of the decarboxylation of substances such as cinnamaldehyde. Other potential sites for nucleophilic attack inhibitors are Cys -152, Asp -28, His- 114, His- 115 and Gln- 477th
The normal catalytic rate of pyruvate decarboxylase is kcat = 10 s -1. In contrast, it is with a Glu -51- Gln mutation to 1.7 s -1.
Mechanism
The enzyme cleaves pyruvate into carbon dioxide and C2 fragment, which is linked to the TPP cofactor. C2 this fragment is bound to the 5-membered ring, which is present as a ylid.