PERTURBATIONS TO THE ACTIVE-SITE OF PHOSPHOTRIESTERASE

Phosphotriesterase catalyzes the hydrolysis of organophosphate nerve a gents. Four amino acid residues, located within the active site pocket , were mutated in an effort to ascertain the roles that these groups p lay in the structure and function of this enzyme. Tryptophan-131 is lo cated at the entrance to the binuclear metal center, and the indole ri ng is positioned to suggest that it could provide a hydrophobic site f or interaction of the aromatic leaving group with optimized substrates . The W131F mutant displays catalytic constants for the hydrolysis of paraoxon that are essentially tile same as those of the wild type enzy me. However, the K-m value for the W131A mutant is elevated by a facto r of 6, consistent with a role for this residue in substrate binding. Aspartate-253 is hydrogen bonded to His-230 which, in turn, is directl y Ligated to the more solvent-exposed metal Ion. The D253N mutant poss esses 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 primar y ligand to the more buried metal ion. Mutation of this residue to his tidine, asparagine, alanine, and cysteine reduces the catalytic activi ty 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 divale nt 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 ca rbamylated and also acts as a bridge between the two divalent cations. Significant losses in catalytic activity are obtained upon mutation o f this residue to either alanine, glutamate, arginine, or methionine. The loss in activity can partially be restored upon inclusion in the a ssay 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 pro pionic acid.