A major objective of the proposed work is to develop new mechanism-based inhibitors for the zinc-containing hydrolytic enzyme carboxypeptidase A (CPA). The approach to be used will be based on earlier work showing that this enzyme can catalyze both enolization and Alpha, Beta -elimination reactions effectively. Preliminary results already show that an appropriately constructed Alpha-cyanoketone reacts with CPA to inactivate the enzyme irreversibly, probably by isomerization of the cyano compound to the corresponding ketenimine, followed by trapping of an active site nucleophile. This proposed pathway will be investigated further by means of kinetic studies, by labeling of the inactivator to determine the stoichiometry of its reaction with the enzyme, and by degradative studies to pinpoint the site of covalent attachment of the inhibitor to CPA. Similar experiments will be performed on Alpha-halo ketoximes which are proposed as possible active site metal ion-activated inhibitors for CPA. In addition, recent experiments have demonstrated that the zinc-containing enzyme angiotensin-converting enzyme (ACE), which is important to blood pressure control and which has been considered to be mechanistically related to CPA, catalyzes Alpha, Beta -elimination with a substrate designed to bind at the active site of ACE and possessing an appropriate leaving group. This observation suggests that mechanism-based or metal ion-activated inhibitors related to those to be prepared for CPA can be designed for ACE. If such inhibitors are successfully employed for ACE, they may not only have important practical applications, as in the control of hypertension, but also the approach used could be applicable to the preparation of mechanism-based inhibitors for other hydrolytic enzymes. Finally, using the Phe-248 variant of CPA, the mechanistic role of the Tyr-248 phenolic hydroxyl in CPA-catalyzed hydrolysis and enolization reactions will be probed. The results obtained should help a great deal in clarifying the possible importance of the phenolic hydroxyl as a proton donor in the action of CPA, a fundamental aspect of the mechansim of catalysis by this enzyme.
| Mobashery, S; Ghosh, S S; Tamura, S Y et al. (1990) Design of an effective mechanism-based inactivator for a zinc protease. Proc Natl Acad Sci U S A 87:578-82 |
| Butler-Ransohoff, J E; Kendall, D A; Kaiser, E T (1988) Use of site-directed mutagenesis to elucidate the role of arginine-166 in the catalytic mechanism of alkaline phosphatase. Proc Natl Acad Sci U S A 85:4276-8 |
