High resolution x-ray crystallographic studies, in combination with detailed kinetic analyses, have yielded a wealth of understanding with respect to enzyme structure and function relationships. The goal of this application is to understand, on a detailed molecular level, the catalytic mechanisms of four enzymatic systems: phosphotriesterase from Pseudomonas diminuta which hydrolyzes certain pesticides and nerve agents, 4-chlorobenzoyl CoA dehalogenase and 4-hydroxybenzoyl CoA thioesterase from Pseudomonas sp. strain CBS-3 which are involved in the pathway that converts 4-chlorobenzoate to 4-hydroxybenzoate, and carbamoyl phosphate synthetase which produces the high energy intermediate carbamoyl phosphate. All of the catalytic mechanisms for these enzymes are believed to proceed through nucleophilic attacks. These enzymes were chosen for investigation due to their interesting biochemical features. The phosphotriesterase utilizes a binuclear metal center for activity while the dehalogenase catalyzes nucleophilic aromatic substitutions. The reaction mechanism of the thioesterase most likely proceeds through a nucleophilic attack but the amino acid sequence of the protein suggests that it is not similar to other thioesterases studied thus far. In the case of carbamoyl phosphate synthetase, the reaction mechanism proceeds via three nucleophilic attacks leading to two reactive intermediates, carboxyphosphate and carbamate. For these proteins, a combination of site-directed mutagenesis experiments and x-ray crystallographic analyses will be employed in order to more fully characterize their three-dimensional architectures, active site geometries, catalytic mechanisms, and substrate specificities.
