THE ROLE OF HIS113 AND HIS114 IN PYRUVATE DECARBOXYLASE FROM ZYMOMONAS-MOBILIS

Pyruvate decarboxylase (PDC) is one of several enzymes that require th iamin diphosphate (ThDP) and a divalent cation as essential cofactors. Recently, the three-dimensional structures of the enzyme from two yea sts have been determined. While these structures shed light on the bin ding of the cofactors and the reaction mechanism, the interactions bet ween the substrate pyruvate and the enzyme remain unclear. We have use d PDC from Zymomonas mobilis as a model for these enzymes in order to study substrate binding. The recombinant enzyme was expressed in Esche richia coli. High yield, simplicity of purification, high stability an d simple kinetics make this model well suited for these studies. Activ ity 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 align ment of all known PDC protein sequences showed two invariant His resid ues (His113 and His114) which, according to the crystal structure of y east 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 cofa ctor and substrate binding. However, the k(cat) is decreased indicatin g that His114, may assist in catalysis. In contrast, substitution of H is113 by Gin renders the enzyme completely inactive. This mutant has d ecreased affinity for both cofactors, as revealed by measurements of t ryptophan 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 ca pable of releasing acetaldehyde from 2-(1-hydroxyethyl)thiamin diphosp hate supplied exogenously. It is proposed that upon substrate binding, His113 is placed close to C2 of the thiazole ring. Subsequent deproto nation of this atom leads to a conformational change that allows a fle xible loop (residues 105-112) that precedes His113 to close over the a ctive site. Hence, replacement of His113 by another residue interferes with this closure of the active site and thus disrupts the catalytic process.