KUKU

KU ScholarWorks

  • myKU
  • Email
  • Enroll & Pay
  • KU Directory
    • Login
    View Item 
    •   KU ScholarWorks
    • Dissertations and Theses
    • Dissertations
    • View Item
    •   KU ScholarWorks
    • Dissertations and Theses
    • Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    The Activity of Phosphorylase Kinase Revisited

    Thumbnail
    View/Open
    Dillon_ku_0099D_15855_DATA_1.pdf (2.099Mb)
    Issue Date
    2018-05-31
    Author
    Dillon, Jackie Ann
    Publisher
    University of Kansas
    Format
    117 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Biochemistry & Molecular Biology
    Rights
    Copyright held by the author.
    Metadata
    Show full item record
    Abstract
    Phosphorylase Kinase (PhK) is an essential regulatory enzyme in the glycogenolysis cascade. PhK is a large, 16-subunit enzyme complex with the subunit stoichiometry (alpha-beta-gamma-delta)4, and is subject to extensive regulation by small molecules and reversible phosphorylation. Because of its large mass of 1.3 MDa, only limited experimental tools are available to study the structure of PhK and elucidate the structural changes that occur within the complex concomitant with activation. Therefore, our understanding of the roles of PhK’s subunits in regulating its kinase activity is woefully incomplete. The goal of this work was to revisit several aspects of PhK’s activity and further explore the roles of the enzyme’s individual subunits. Chemical crosslinking was the principal technique used in these studies because it is well-suited to study large, multi-subunit complexes. Using chemical crosslinking, plus additional methods, three major discoveries are presented regarding PhK’s activation and substrate recognition. First, zero-length oxidative crosslinking was used to selectively study conformational changes in the regulatory beta subunits, which led to the formulation of a model for activation of PhK. The model proposes that modification of the N-terminus of beta is key to activation of the catalytic gamma subunit and to conformational changes in the beta subunits, which likely cause global structural changes in the complex. Secondly, a two-step crosslinking approach revealed novel interactions between the regulatory alpha and beta subunits and PhK’s substrate, glycogen phosphorylase, establishing the first direct evidence of substrate binding sites on the regulatory subunits of the enzyme. Lastly, a temperature-dependent conformational change in the beta and gamma subunits was discovered to occur between the standard assay temperature of 30 °C and the physiological temperature of 40 °C. This temperature-dependent conformational change coincides with a surprising activation of PhK at physiological temperature. Steady progress continues to be made in studying the PhK complex. While the picture is still far from complete, this work contributes important details regarding the alpha, beta, and gamma subunits and their expanded roles in PhK’s activation and substrate interaction. Further exploration of the structure and activity of PhK is necessary to fully understand its critical function in glycogenolysis.
    URI
    http://hdl.handle.net/1808/27071
    Collections
    • Molecular Biosciences Dissertations and Theses [273]
    • Dissertations [4473]

    Items in KU ScholarWorks are protected by copyright, with all rights reserved, unless otherwise indicated.


    We want to hear from you! Please share your stories about how Open Access to this item benefits YOU.


    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

    Browse

    All of KU ScholarWorksCommunities & CollectionsThis Collection

    My Account

    LoginRegister

    Statistics

    View Usage Statistics

    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

    The University of Kansas
      Contact KU ScholarWorks
    Lawrence, KS | Maps
     
    • Academics
    • Admission
    • Alumni
    • Athletics
    • Campuses
    • Giving
    • Jobs

    The University of Kansas prohibits discrimination on the basis of race, color, ethnicity, religion, sex, national origin, age, ancestry, disability, status as a veteran, sexual orientation, marital status, parental status, gender identity, gender expression and genetic information in the University’s programs and activities. The following person has been designated to handle inquiries regarding the non-discrimination policies: Director of the Office of Institutional Opportunity and Access, IOA@ku.edu, 1246 W. Campus Road, Room 153A, Lawrence, KS, 66045, (785)864-6414, 711 TTY.

     Contact KU
    Lawrence, KS | Maps