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Kinetic Analysis of the Processivity of Human Cytochrome P450 17A1
Author(s) -
Gonzalez Eric,
Nagy Leslie,
Guengerich Frederick
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.722.13
Subject(s) - pregnenolone , processivity , chemistry , cytochrome p450 , cyp17a1 , hydroxylation , enzyme , biochemistry , steroid , hormone , polymerase
Human cytochrome P450 (P450) 17A1 is one of several P450 monoxygenases involved in the biosynthesis of steroids. Notably, P450 17A1 is responsible for 17α‐hydroxylation of the 21‐carbon steroids pregnenolone and progesterone, which are used in glucocorticoid production. 17α‐Hydroxypregnenolone and 17α‐hydroxyprogesterone are also further oxidized by P450 17A1 in a 17α,20‐lyase reaction generating the 19‐carbon androgens dehydroepiandrosterone and androstenendione, respectively. Selective stimulation of the 17α,20‐lyase reaction is observed with human cytochrome b 5 ( b 5 ). Inhibition of P450 17A1 is a goal in some cancer therapies because of its role in generating sex hormone precursors through the cleavage reaction. However, complete inhibition of the enzyme concomitantly impedes glucocorticoid synthesis. This issue poses the question of whether the 17α,20‐lyase reaction succeeds 17α‐hydroxylation in a successive or distributive manner. To date, the processivity of human P450 17A1 (with and without b 5 ) has yet to be systematically determined. Steady‐state kinetic parameters have been measured for human P450 17A1 reactions and binding constants have been obtained via spectral titrations. Pulse‐chase assays using radio‐labeled substrates and pre‐steady‐state analyses further elucidate the rate constants of key steps in the two‐reaction mechanism for both steroids. Human P450 17A1 is partially processive with pregnenolone. The kinetic analyses yield data to further model the processivity in the presence and absence of b 5 utilizing KinTek Explorer® software.