Abstract
Incubation of phenyldiazene (PhN = NH) with lanosterol 14 alpha-demethylase, a cytochrome P-450 enzyme (CYP51) that oxidatively removes the 14 alpha-methyl group of lanosterol, results in the appearance of a 478-nm band indicative of phenyl-iron complex formation. In situ oxidation of the phenyl-iron complex by ferricyanide yields exclusively the N-phenylprotoporphyrin IX regioisomer with the phenyl group on the nitrogen of pyrrole ring C (NC). The biphenyl-iron complex formed in the analogous reaction of the enzyme with biphenyldiazene similarly rearranges on treatment with ferricyanide to the NC regioisomer of N-biphenylprotoporphyrin IX. The active site cavity must therefore be at least 10 A high directly above the iron atom and pyrrole ring C of the heme group, and lanosterol binds to the enzyme in the region above pyrrole ring C. Phenyl-iron complex formation is not detected spectroscopically with cytochrome P-450SG1, a catalytically inactive G310D mutant of lanosterol 14 alpha-demethylase in which the sixth iron coordination site is thought to be occupied by an imidazole ligand. Nevertheless, oxidation of the phenyldiazene-treated enzyme with ferricyanide provides the NA and NC regioisomers of N-phenylprotoporphyrin IX in a 40:60 ratio. The single amino acid substitution in cytochrome P-450SG1 thus causes a conformational change that retracts the amino acid residues that cover pyrrole ring A and moves an imidazole ligand into the active site.
Highlights
Incubation of phenyldiazene (PhN = NH) with lanosterol 14a-demethylase,a cytochrome P-450 enzyme (CYP51) that oxidativelyremovesthe14a-methyl group of lanosterol, results in the appearanocfea 478
Thebiphenyl-iron complex formed in the analogous reaction of the en- an enzyme that has been purified from Saccharomyces cerezymewithbiphenyldiazenesimilarly rearranges on treatment with ferricyanide to the NCregioisomer of
We report here application of this method to analysiosf the active sitetopologies of spectra and retention times, including coelution, with those of the authentic N-aryl PPIX isomers
Summary
The active site cavity must be at least 10 A high directly above the iron atom and pyrrole ringC of the heme group, and lanosterol binds to the enzyme in the region above uisiae (I),Candida albicans [2], rat liver [3],and pig liver [4]. Incubation of treated enzyme with ferricyanide results in the isolation, in relatively low yield (-20% of normal), of a 40:60 ratio of the NA and Nc regioisomers of N-phenyl PPIX (Fig. 3).
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