RESUMO
Mammalian adrenodoxin (Adx) has been known for many years as an essential electron mediator in mitochondrial cytochrome P450 systems. Because of its ability to support several cytochrome P450 enzymes, it is involved not only in adrenal steroid hormone biosynthesis but also in vitamin D and bile acid metabolism. Recently, Adx is increasingly gaining attention because of its potential for pharmaceutical industry and biotechnology. With human cytochromes P450 becoming important drug targets, suitable Adx-based screening systems have to be developed to test putative new drugs. Moreover, in artificial systems, Adx has been shown to functionally interact with diverse bacterial cytochromes P450 catalyzing a variety of chemically interesting reactions. Putative biotechnological applications of such Adx-containing reconstituted systems are discussed.
Assuntos
Adrenodoxina/fisiologia , Ferredoxinas/fisiologia , Adrenodoxina/biossíntese , Adrenodoxina/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Coenzimas/biossíntese , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/fisiologia , Avaliação Pré-Clínica de Medicamentos , Ferredoxinas/biossíntese , Ferredoxinas/química , Humanos , Mitocôndrias/enzimologia , Oxirredução , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.