Kinetic mechanism of the type II calmodulin-dependent protein kinase: studies of the forward and reverse reactions and observation of apparent rapid-equilibrium ordered binding.
The kinetic reaction mechanism of the type II calmodulin-dependent protein kinase was studied by using its constitutively active kinase domain. Lacking regulatory features, the catalytic domain simplified data collection, analysis, and interpretation. To further facilitate this study, a synthetic peptide was used as the kinase substrate. Initial velocity measurements of the forward reaction were consistent with a sequential mechanism. The patterns of product and dead-end inhibition studies best fit an ordered Bi Bi kinetic mechanism with ATP binding first to the enzyme, followed by binding of the peptide substrate. Initial-rate patterns of the reverse reaction of the kinase suggested a rapid-equilibrium mechanism with obligatory ordered binding of ADP prior to the phosphopeptide substrate; however, this apparent rapid-equilibrium ordered mechanism was contrary to the observed inhibition by the phosphopeptide which is not supposed to bind to the kinase in the absence of ADP. Inspection of product inhibition patterns of the phosphopeptide with both ATP and peptide revealed that an ordered Bi Bi mechanism can show initial-rate patterns of a rapid-equilibrium ordered system when a Michaelis constant for phosphopeptide, Kip, is large relative to the concentration of phosphopeptide used. Thus, the results of this study show an ordered Bi Bi mechanism with nucleotide binding first in both directions of the kinase reaction. All the kinetic constants in the forward and reverse directions and the Keq of the kinase reaction are reported herein. To provide theoretical bases and diagnostic aid for mechanisms that can give rise to typical rapid-equilibrium ordered kinetic patterns, a discussion on various sequential cases is presented in the Appendix.
Kwiatkowski
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AP
Department of Chemistry, Ohio State University, Columbus 43210.
Huang
C Y
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King
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MM
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; C063391:syntide-2; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases