title: Photoaffinity labeling of methyl farnesoate epoxidase in cockroach corpora allata.
abstract: The last enzyme in the biosynthetic pathway to juvenile hormone III in the corpora allata of hemimetabolous insects is methyl farnesoate epoxidase, a cytochrome P450 monooxygenase. Assays with intact glands incubated in vitro and with gland homogenates have identified a series of 1,5-disubstituted imidazoles as potent inhibitors of the enzyme. We have designed, synthesized and tested two imidazoles, diazirine-Ice T and benzophenone-Ice T, in which a radiolabeled and photoactivatable diazirine or benzophenone group was introduced to label the hydrophobic substrate binding site of the enzyme. Our results show that these bifunctional compounds inhibit JH III synthesis by intact glands as well as methyl farnesoate epoxidation by gland homogenates. Moreover both compounds selectively label a protein of ca. 55 kDa in corpora allata of the cockroach, Diploptera punctata. These photoaffinity labels, which use an imidazole to coordinate to the heme iron and a photoreactive group to modify the hydrophobic substrate binding pocket, are specific and effective probes for the molecular analysis of methyl farnesoate epoxidase.
authors.affiliation: Department of Entomology, University of Arizona, Tucson 85721, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Andersen
authors.forename: M
authors.initials: M
authors.lastname: Ceruso
authors.forename: G C
authors.initials: GC
authors.lastname: Unnithan
authors.forename: E
authors.initials: E
authors.lastname: Kuwano
authors.forename: G D
authors.initials: GD
authors.lastname: Prestwich
authors.forename: R
authors.initials: R
authors.lastname: Feyereisen
mesh_terms: D000345:Affinity Labels / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001389:Azirines / Q000737:chemistry / Q000494:pharmacology; D001577:Benzophenones / Q000737:chemistry / Q000494:pharmacology; D003058:Cockroaches / Q000201:enzymology*; D003335:Corpora Allata / Q000201:enzymology*; D005260:Female; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology; D066298:In Vitro Techniques; D007605:Juvenile Hormones / Q000096:biosynthesis; D015394:Molecular Structure; D010105:Oxygenases / Q000032:analysis / Q000096:biosynthesis
chemical_list: D000345:Affinity Labels; D001389:Azirines; D001577:Benzophenones; D007093:Imidazoles; D007605:Juvenile Hormones; C094895:benzophenone-Ice T; C094896:diazirine-Ice T; D010105:Oxygenases; C032432:methyl farnesoate epoxidase