title: Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17.
abstract: Cytochrome P450c17 (CYP17) catalyzes both the 17alpha-hydroxylase and 17,20-lyase reactions in human steroid biosynthesis. Cytochrome b5 (b5) stimulates the rate of the 17,20-lyase reaction 10-fold with little influence on 17alpha-hydroxylase activity. Studies with apo-b5 suggest that stimulation of 17,20-lyase activity results from an allosteric action on the hCYP17 x POR complex, rather than electron transfer by b5. We hypothesized that specific residues on b5 interact with the hCYP17 x POR complex and that targeted mutation of surface-exposed residues might identify b5 residues critical for stimulating 17,20-lyase activity. We constructed, expressed, and purified 14 single plus 3 double b5 mutations and assayed their ability to stimulate 17,20-lyase activity. Most mutations did not alter the capacity of b5 to stimulate 17,20-lyase activity or appeared to modestly alter the affinity of b5 for the hCYP17 x POR complex. In contrast, mutation of E48, E49, or R52 reduced the maximal stimulation of 17,20-lyase activity. In particular, b5 mutation E48G + E49G lost over 95% of the capacity to stimulate 17,20-lyase activity, yet this mutation retained normal electron transfer properties. In addition, mutation E48G + E49G did not impair stimulation of 17,20-lyase activity by wild-type b5, suggesting that the mutation binds poorly to the site of the hCYP17 x POR complex occupied by b5. These data suggest that a specific allosteric binding site on b5, which includes residues E48, E49, and possibly R52, mediates the stimulation of 17,20-lyase activity.
authors.affiliation: Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857, USA.
authors.forename: Jacqueline L
authors.initials: JL
authors.lastname: Naffin-Olivos
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Auchus
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008190:Lyases / Q000378:metabolism*; D008958:Models, Molecular; D017354:Point Mutation; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D018698:Glutamic Acid; D015786:Cytochromes b5; D013254:Steroid 17-alpha-Hydroxylase; D008190:Lyases