The long term goal of this proposal is to define the proteolytic pathway which degrades CYP2El and to identify those regions of the protein that are important for targeting the enzyme for degradation. The results of this proposal will lead a better understanding in the role of structural motifs in the degradation of CYP2El. The goal of this project will be accomplished through the direct determination of the steady state half- life of the native enzyme, amino- and carboxyl-terminal deleted mutants and chimeric proteins in which regions of the longer half-life CYP2Bl are combined with those of CYP2El. Once a region is defined through analysis of chimeric enzymes, specific site directed mutants will be prepared in order to identify residues which may be important in the determination of the degradative path of the enzyme. In all cases, half-life studies will be coupled with the use of selective inhibitors of key intracellular proteolytic enzymes in order to determine whether chimeric enzymes follow the same degradative path as the native protein. The susceptibility of the various constructs to proteolysis i,i vitro will also be determined.
