The long-term objectives of this proposal are directed at an understanding of the relationship between structure and function biological systems. This application is targeted, in part, at carbamoyl phosphate synthetase (CPS), an enzyme that is responsible for the production of the key intermediate during the biosynthesis of arginine, detoxification of ammonia, and the formation of pyrimidine nucleotides. The synthesis of carbamoyl-P by a single protein is one of the most complicated reactions in biological chemistry since five different reaction products are formed in a reaction sequence that required four chemical events involving three unstable intermediates. This remarkable transformation occurs through the coordinated efforts of three different active sites that are separated by nearly 100A! Two of the three chemical intermediates must be translocated through a molecular tunnel connection the three active sites with on another.
The specific aims of this proposal include the following: (1) The mechanism for the synchronization of the key chemical events with the migration of reaction inter- mediates will be elucidated using biophysical probes. (2) The molecular tunnel within the interior of CPS will be structurally modified with the aim of retarding or impeding the passage of chemical intermediates from one active site to another. These studies will quantitate the degree of rate limitation imparted by this elusive event and determine whether the molecular tunnel serves as the primary conduit for signal transduction between consecutive active sites. (3) The catalytic activity of CPS is tightly regulated by the concentrations of metabolic intermediates. Structural and kinetic investigations will be used to identify the conformational changes that are propagated by the binding of the allosteric ligands to the protein. (4) The amidotransferase family of enzymes has evolved to serve as efficient molecular machines for the production and delivery of ammonia to a distal active site. Structural and mechanistic probes will be applied to members of this superfamily to ascertain the similarities and differences in the strategies employed by Nature for the channeling of metabolic intermediate from one sit to another.
Showing the most recent 10 out of 40 publications
