Perturbations to the active site of phosphotriesterase.
Phosphotriesterase catalyzes the hydrolysis of organophosphate nerve agents. Four amino acid residues, located within the active site pocket, were mutated in an effort to ascertain the roles that these groups play in the structure and function of this enzyme. Tryptophan-131 is located at the entrance to the binuclear metal center, and the indole ring is positioned to suggest that it could provide a hydrophobic site for interaction of the aromatic leaving group with optimized substrates. The W131F mutant displays catalytic constants for the hydrolysis of paraoxon that are essentially the same as those of the wild type enzyme. However, the Km value for the W131A mutant is elevated by a factor of 6, consistent with a role for this residue in substrate binding. Aspartate-253 is hydrogen bonded to His-230 which, in turn, is directly ligated to the more solvent-exposed metal ion. The D253N mutant possesses catalytic constants that are virtually the same as those of the wild type enzyme, while the D253A mutant is reduced in activity by 500-fold. These results are consistent with a model where this residue is required to orientate the imidazole side chain of His-230 for proper interaction with the binuclear metal center. Aspartate-301 is a primary ligand to the more buried metal ion. Mutation of this residue to histidine, asparagine, alanine, and cysteine reduces the catalytic activity by factors of 2.6 x 10(4), 2.7 x 10(3), 5.6 x 10(2), and 1.5 x 10(2), respectively. These results indicate that alterations to the direct metal ligands, even with residues that can strongly coordinate divalent cations, cause a severe disruption to the proper functioning of the active site. In the wild type enzyme, the side chain of Lys-169 is carbamylated and also acts as a bridge between the two divalent cations. Significant losses in catalytic activity are obtained upon mutation of this residue to either alanine, glutamate, arginine, or methionine. The loss in activity can partially be restored upon inclusion in the assay mixture of short-chain carboxylic acids. A 25-fold enhancement in k(cat) is observed for the K169A mutant in the presence of 100 mM propionic acid.
Kuo
J M
JM
Department of Chemistry, Texas A&M University, College Station 77843, USA.
Chae
M Y
MY
Raushel
F M
FM
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043303:Aryldialkylphosphatase; D001665:Binding Sites / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D013329:Structure-Activity Relationship
D000596:Amino Acids; D004950:Esterases; D043303:Aryldialkylphosphatase