The structural basis of substrate activation in yeast pyruvate decarboxylase - A crystallographic and kinetic study

The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain w ith the activator pyruvamide has been determined to 2.4 Angstrom resolution . The asymmetric unit of the crystal contains two subunits, and the tetrame ric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the acti ve site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vi cinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the su bunit in the closed conformation, about 10 Angstrom away from residue C221. This second pyruvamide molecule might function in stabilizing the unique o rientation of the R domain in this subunit which in turn is important for d imer-dimer interactions in the activated tetramer. No difference electron d ensity in the close vicinity of the side chain of residue C221 was found, i ndicating that this residue does not form a covalent adduct with an activat or molecule. Kinetic experiments showed that substrate activation was not a ffected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event i n the activation process triggered by the activator pyruvamide and that cov alent modification of C221 is not required for this transition to occur. Ba sed on these findings, a possible mechanism for the activation of PDC by it s substrate, pyruvate, is proposed.