Selective covalent labeling with radiolabeled suicide substrates for isolating P450s

Publisher Summary This chapter describes the use of covalent tagging technique to isolate a target P450 protein, the obtainment of specific peptide sequences, and the subsequent cloning of the corresponding complementary deoxyribonucleic acid (cDNA). Mechanism-based enzyme inactivators are irreversible inactivators of specific target enzymes that catalyze their own destruction by unmasking a latent functional group during the catalytic cycle of the enzyme. The reactive intermediate thus formed may either evolve to a normal reaction product, which is then released from the enzyme, or become covalently attached. It is the ratio between these two competing reactions that decides primarily the rate of enzyme inactivation. A promising route is offered by introducing a substrate-specific step into the separation procedure. This may be achieved by using so-called “suicide substrates” really mechanism-based inhibitors) to introduce a covalent tag into the target enzyme and to allow tracking it during separation. As an example of the power of this approach and its shortcomings, this chapter describes the isolation of the first plant fatty acid ω-hydroxylase and its subsequent cloning.