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. [4]
Active site residues
Each active site has 20 amino acid residues, including the acidic Glu-477, which interacts with the TPP ring, and Glu-51, which participates with the binding of the cofactor. These glutamates also stabilize the ylid of TPP, acting as proton donors. The nonpolar environment around this Glu 477 is nonpolar, which contributes to a higher than normal pKa (normal Glu and Asp pKa's are around 4.6 in small proteins). [5]
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 ylide. [4] 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 substrate-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. [4]
Residues Asp-444 and Asp-28 bind Mg2+. 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 availability. If the substrate bound in the active site is pyruvate, the enzyme is activated by a conformational change in this regulatory site. [6] 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. [6]
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. [4]