Mechanism-based inactivators as probes of cytochrome P450 structure and function.
The cytochromes P450 superfamily of enzymes is a group of hemeproteins that catalyze the metabolism of an extensive series of compounds including drugs, chemical carcinogens, fatty acids, and steroids. They oxidize substrates ranging in size from ethylene to cyclosporin. Although significant efforts have been made to obtain structural information on the active sites of the microbial P450s, relatively little is currently known regarding the identities of the critical amino acid residues in the P450 active sites that are involved in substrate binding and catalysis. Since information on the crystal structures of the eukaryotic P450s has been relatively limited, investigators have used a variety of other techniques in attempts to elucide the structural features that play a role in the catalytic properties and substrate specificity at the enzyme active site. These include site-directed mutagenesis, natural mutations, homology modeling, mapping with aryl-iron complexes, affinity and photoaffinity labeling, and mechanism-based inactivators. A variety of different mechanism-based inactivators have proven to be useful in identifiying active site amino acid residues involved in substrate binding and catalysis. In this review we present a sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins. Labeled peptides isolated from the inactivated proteins have been analyzed by N-terminal amino acid sequencing in conjunction with mass spectrometry to determine the sites of covalent modification. Mechanistic studies aimed at identifying the basis for the inactivation following adduct formation are also presented.
Kent
U M
UM
Department of Pharmacology, University of Michigan, Ann Arbor 48109-0632, USA.
Juschyshyn
M I
MI
Hollenberg
P F
PF
D000595:Amino Acid Sequence; D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System