title: Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation.
abstract: The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg(2+) binds first to the M1 site as a complex with ATP and is followed by Mg(2+) binding to the M2 site. Concurrently, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. Lastly, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.
authors.affiliation: Biology & Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. Electronic address: amitdas@barc.gov.in.
authors.forename: Amit
authors.initials: A
authors.lastname: Das
authors.affiliation: Biology & Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
authors.forename: Oksana
authors.initials: O
authors.lastname: Gerlits
authors.affiliation: UT/ORNL Center for Molecular Biophysics, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
authors.forename: Jerry M
authors.initials: JM
authors.lastname: Parks
authors.affiliation: Biology & Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Langan
authors.affiliation: Biology & Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
authors.forename: Andrey
authors.initials: A
authors.lastname: Kovalevsky
authors.affiliation: Biology & Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. Electronic address: hellerwt@ornl.gov.
authors.forename: William T
authors.initials: WT
authors.lastname: Heller
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium