Kinetic Processivity of the Two-Step Oxidations of Progesterone and Pregnenolone to Androgens by Human Cytochrome P450 17A1

J Biol Chem. 2017 Aug 11;292(32):13168-13185. doi: 10.1074/jbc.M117.794917. Epub 2017 Jul 6.

Abstract

Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome b5 (b5), but the exact role of b5 in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. b5 did not enhance reaction rates by decreasing the koff rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse-chase experiments. (S)-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC50 values for (S)-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, i.e. some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography.

Keywords: cytochrome P450; enzyme kinetics; oxidation-reduction (redox); pre-steady-state kinetics; steady-state kinetics; steroid metabolism; steroidogenesis.

Publication types

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

MeSH terms

  • 17-alpha-Hydroxypregnenolone / chemistry
  • 17-alpha-Hydroxypregnenolone / metabolism*
  • Androstenedione / chemistry
  • Androstenedione / metabolism
  • Animals
  • Binding Sites
  • Biocatalysis / drug effects
  • Cytochrome P-450 Enzyme Inhibitors / chemistry
  • Cytochrome P-450 Enzyme Inhibitors / metabolism
  • Cytochrome P-450 Enzyme Inhibitors / pharmacology
  • Cytochromes b5 / genetics
  • Cytochromes b5 / metabolism*
  • Dehydroepiandrosterone / chemistry
  • Dehydroepiandrosterone / metabolism*
  • Humans
  • Imidazoles / chemistry
  • Imidazoles / metabolism
  • Imidazoles / pharmacology
  • Kinetics
  • Ligands
  • Models, Molecular*
  • NADPH-Ferrihemoprotein Reductase / genetics
  • NADPH-Ferrihemoprotein Reductase / metabolism*
  • Naphthalenes / chemistry
  • Naphthalenes / metabolism
  • Naphthalenes / pharmacology
  • Oxidation-Reduction
  • Pregnenolone / chemistry
  • Pregnenolone / metabolism*
  • Progesterone / chemistry
  • Progesterone / metabolism
  • Protein Conformation
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Stereoisomerism
  • Steroid 17-alpha-Hydroxylase / antagonists & inhibitors
  • Steroid 17-alpha-Hydroxylase / chemistry
  • Steroid 17-alpha-Hydroxylase / genetics
  • Steroid 17-alpha-Hydroxylase / metabolism*

Substances

  • CYB5A protein, human
  • Cytochrome P-450 Enzyme Inhibitors
  • Imidazoles
  • Ligands
  • Naphthalenes
  • Recombinant Proteins
  • 17-alpha-Hydroxypregnenolone
  • Androstenedione
  • Dehydroepiandrosterone
  • Progesterone
  • Pregnenolone
  • Cytochromes b5
  • CYP17A1 protein, human
  • Steroid 17-alpha-Hydroxylase
  • NADPH-Ferrihemoprotein Reductase
  • orteronel