Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a divalent cation as essential cofactors. Recently, the three-dimensional structures of the enzyme from two yeasts have been determined. While these structures shed light on the binding of the cofactors and the reaction mechanism, the interactions between the substrate pyruvate and the enzyme remain unclear. We have used PDC from Zymomonas mobilis as a model for these enzymes in order to study substrate binding. The recombinant enzyme was expressed in Escherichia coli. High yield, simplicity of purification, high stability and simple kinetics make this model well suited for these studies. Activity measurements in the pH range between 5.8 and 8.5 indicated that a His residue may be involved in substrate binding. Analysis of an alignment of all known PDC protein sequences showed two invariant His residues (His113 and His114) which, according to the crystal structure of yeast PDC, are in the vicinity of the active site. Here we demonstrate that replacement of His114 by Gin does not have a great effect on cofactor and substrate binding. However, the kcat is decreased indicating that His114, may assist in catalysis. In contrast, substitution of His113 by Gin renders the enzyme completely inactive. This mutant has decreased affinity for both cofactors, as revealed by measurements of tryptophan fluorescence quenching. However, this decreased affinity is insufficient to account for the complete loss of activity. Despite its inability to support overall catalysis, this [Gln113]PDC mutant is capable of releasing acetaldehyde from 2-(1-hydroxyethyl)thiamin diphosphate supplied exogenously. It is proposed that upon substrate binding, His113 is placed close to C-2 of the thiazole ring. Subsequent deprotonation of this atom leads to a conformational change that allows a flexible loop (residues 105-112) that precedes His113 to close over the active site. Hence, replacement of His113 by another residue interferes with this closure of the active site and thus disrupts the catalytic process.
Department of Chemistry
University of Cambridge