Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17

Biochemistry. 2006 Jan 24;45(3):755-62. doi: 10.1021/bi051623y.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Cell Line
  • Cytochromes b5 / chemistry*
  • Cytochromes b5 / genetics
  • Cytochromes b5 / metabolism*
  • Glutamic Acid / genetics
  • Glutamic Acid / metabolism*
  • Humans
  • Lyases / metabolism*
  • Models, Molecular
  • Point Mutation
  • Steroid 17-alpha-Hydroxylase / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity


  • Glutamic Acid
  • Cytochromes b5
  • Steroid 17-alpha-Hydroxylase
  • Lyases