Texas A&M University LibrariesTexas A&M University LibrariesTexas A&M University Libraries
    • Help
    • Login
    OAKTrust
    View Item 
    •   OAKTrust Home
    • Colleges and Schools
    • Office of Graduate and Professional Studies
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    • View Item
    •   OAKTrust Home
    • Colleges and Schools
    • Office of Graduate and Professional Studies
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Investigations on the Mechanism of Allosteric Activtion of Rabbit Muscle Glycogen Phosphorylase b by AMP

    Thumbnail
    View/ Open
    BIGLEY-DISSERTATION.pdf (1.121Mb)
    Date
    2010-07-14
    Author
    Bigley, Andrew N.
    Metadata
    Show full item record
    Abstract
    Much work has been carried out on glycogen phosphorylase over the last seventy years. Interest has persisted due not only to the usefulness of phosphorylase as a model system of allostery, but also due to the connection to the disease state in type II diabetes. The bulk of research consists of structural studies utilizing the wild-type enzyme from rabbit muscle. In this study we have employed linkage analysis in combination with structural perturbations via site-directed mutagenesis to test kinetic models of activation of phosphorylase b by AMP, and to examine the roles of the N-terminus, the acidic patch, ?-helix 1 and the 280?s loop in activation by AMP. Experiments have been carried out on purified glycogen phosphorylase b variants to determine the effects of perturbations in vitro. The kinetic models of activation by AMP are found to be a relatively accurate description of kinetic behavior of wild-type phosphorylase b, but are found to be technically incorrect with respect to the absolute requirements of two equivalents of AMP to be bound prior to catalysis. Phosphorylase b demonstrates activity in the absence of AMP, though only at high concentrations of phosphate, and a hybrid phosphorylase b with only a single functional AMP binding sight shows slight activation. The truncate ?2-17 shows weakened binding to AMP and phosphate in the apo enzyme, but maintains activation by AMP to an affinity similar to that of wild-type, indicating that the N-terminus is not required for activation by AMP, but has a role in establishing the affinity for both AMP and phosphate in the apo enzyme. Perturbations of the acidic patch indicate that interactions between the acidic patch and the N-terminus enhance the affinities in the apo enzyme, suggesting that the structures of the N-terminus at the acidic patch may represent an active form of the enzyme. ?-helix 1 is found to have a role in homotropic cooperativity in phosphorylase b, but not in heterotropic activation by AMP, while the 280?s loop is confirmed to have a role in the heterotropic coupling between AMP and phosphate. Based on the findings in this study an alternate structural model of activation by AMP involving ?-helix 8 is proposed.
    URI
    https://hdl.handle.net/1969.1/ETD-TAMU-2009-05-483
    Subject
    Allostery, Regulation, Glycogen Phosphorylase, AMP
    Collections
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    Citation
    Bigley, Andrew N. (2009). Investigations on the Mechanism of Allosteric Activtion of Rabbit Muscle Glycogen Phosphorylase b by AMP. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /ETD -TAMU -2009 -05 -483.

    DSpace software copyright © 2002-2016  DuraSpace
    Contact Us | Send Feedback
    Theme by 
    Atmire NV
     

     

    Advanced Search

    Browse

    All of OAKTrustCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsDepartmentTypeThis CollectionBy Issue DateAuthorsTitlesSubjectsDepartmentType

    My Account

    LoginRegister

    Statistics

    View Usage Statistics
    Help and Documentation

    DSpace software copyright © 2002-2016  DuraSpace
    Contact Us | Send Feedback
    Theme by 
    Atmire NV