The catalytic (C) subunit of cAMP-dependent protein kinase (PKA) can be regulated by co- and post-translational modifications. The C-subunit is cotranslationally N-myristylated. N-myristylation is catalyzed by the enzyme N-myristyl transferase (NMT), and it is the incorporation of the 14 carbon, saturated fatty acid, myristic acid, onto the N-terminal glycine residue of a target protein. In PKA, Nmyristylation enhances the thermal stability of the protein and increases membrane binding in RII but not RI holoenzyme complexes. In these studies, the myristylated Csubunit was crystallized in different states including bound to a substrate peptide alone as well as to a substrate peptide and an ATP analogue. Furthermore, time-resolved fluorescence anisotropy and MD simulations were performed to analyze the effects of N-myristylation on C-subunit dynamics. These studies showed that N-myristylation stabilizes the N-terminus and myristate pocket of PKA and exhibits effects on dynamics throughout the enzyme including at the active site. Myristylation may, therefore, provide allosteric regulation of PKA.
The reaction mechanism of PKA is also well-characterized. ADP-release is the rate-limiting step in PKA reaction turnover, and ADP-release is governed by magnesium concentration. PKA binds two magnesium ions termed Mg1 and Mg2 with the numbering based on the order in which the ions were thought to bind. However, recent studies suggest that the magnesium ions may bind in reverse order, and the mode of magnesium and ADP-release following phosphoryl transfer is unresolved. In these studies, the C-subunit was crystallized displaying partial and complete phosphoryl transfer of AMP-PNP onto a substrate peptide. These structures showed that Mg1 is released following complete phosphoryl transfer, and therefore, Mg1 release may be an important step preceding ADP release. Also, the C-subunit of PKA was crystallized in an apo state and bound to ADP. The ADP bound structure showed that ADP binds to the C-subunit with one magnesium ion, and this ion corresponds to Mg2 verifying the results from the partial and complete phosphoryl transfer structures. Furthermore, the ADP bound structure adopts a novel conformation that may represent the possible motions associated with ADP release including opening of the Gly-rich loop and C-tail away from the active site.
|Advisor:||Taylor, Susan S.|
|Commitee:||Handel, Tracy, Komives, Elizabeth, Newton, Alexandra, Trejo, JoAnn|
|School:||University of California, San Diego|
|School Location:||United States -- California|
|Source:||DAI-B 75/08(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Biochemistry, Biophysics|
|Keywords:||Camp-dependent protein kinase, Myristoylation, Myristylation, Phosphorylation, Protein kinase a, Reaction progression|
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