• About
    • Login
    View Item 
    •   Institutional Repository Home
    • Electronic Theses and Dissertations
    • Electronic Theses and Dissertations
    • View Item
    •   Institutional Repository Home
    • Electronic Theses and Dissertations
    • Electronic Theses and Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of Institutional RepositoryCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsDepartmentThis CollectionBy Issue DateAuthorsTitlesSubjectsDepartment

    My Account

    LoginRegister

    The Kinetic and Chemical Mechanisms of Human Cytochrome P450 17A1

    Gonzalez, Eric
    : https://etd.library.vanderbilt.edu/etd-04102017-150657
    http://hdl.handle.net/1803/12107
    : 2017-04-10

    Abstract

    Human cytochrome P450 (P450) 17A1 is an essential enzyme in the steroid biosynthesis pathway that mediates a critical branch point which leads to either glucocorticoid or sex hormone production. P450 17A1 is a bifunctional enzyme that catalyzes 17α-hydroxylation of pregnenolone and progesterone and subsequently a 17,20-lyase reaction that generates dehydroepiandrosterone and androstenedione, respectively. While the 17α-hydroxy steroids are substrates for the androgen generating cleavage reaction, they are also precursors to glucocorticoids. Selective inhibition of the 17,20-lyase reaction to reduce androgen levels, without disrupting glucocorticoid production, is a current goal in prostate cancer therapy. The objective of this investigation was to evaluate the chemical and kinetic mechanisms of human P450 17A1 that enable catalysis of two distinct reactions, with the purpose of contributing greater insight for the development of cleavage-selective inhibitors. The chemical mechanism of the 17,20-lyase reaction was assessed and the results provide compelling support for a Compound I-mediated mechanism but have not ruled out a ferric peroxide mechanism. Novel hydroxylation products were identified in the course of these studies. The majority of the new products were 16-hydroxy steroids and, remarkably, hydroxylation of a B-ring carbon was also observed (6β,16α,17α-trihydroxyprogesterone). One critical factor for reaction-selective inhibition of P450 17A1 is the processivity in catalyzing the sequential reactions. The kinetic mechanism of human P450 17A1 was evaluated using steady-state and pre-steady-state methods, and a minimal kinetic model was developed using KinTek Explorer® software. Human P450 17A1 was primarily distributive in catalyzing the two reactions but was more processive with pregnenolone than with progesterone. Furthermore, cytochrome b5 (b5) enhanced the processivity and the results indicate that it functions as a shunt towards sex hormone production. A theoretical model was proposed that includes multiple enzyme conformations, including a more efficient, processive conformation that may be stabilized by b5. As such, selective inhibition of the 17,20-lyase reaction is complicated by the propensity of human P450 17A1 to catalyze the sequential reactions processively when b5 is present, and it is possible that it may only be accomplished by disrupting the interactions between the two proteins.
    Show full item record

    Files in this item

    Icon
    Name:
    Gonzalez.pdf
    Size:
    8.383Mb
    Format:
    PDF
    View/Open

    This item appears in the following collection(s):

    • Electronic Theses and Dissertations

    Connect with Vanderbilt Libraries

    Your Vanderbilt

    • Alumni
    • Current Students
    • Faculty & Staff
    • International Students
    • Media
    • Parents & Family
    • Prospective Students
    • Researchers
    • Sports Fans
    • Visitors & Neighbors

    Support the Jean and Alexander Heard Libraries

    Support the Library...Give Now

    Gifts to the Libraries support the learning and research needs of the entire Vanderbilt community. Learn more about giving to the Libraries.

    Become a Friend of the Libraries

    Quick Links

    • Hours
    • About
    • Employment
    • Staff Directory
    • Accessibility Services
    • Contact
    • Vanderbilt Home
    • Privacy Policy