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J Biol Chem. 1997 Dec 12;272(50):31235-40.

Characterization of substrate phosphorylation and use of calmodulin mutants to address implications from the enzyme crystal structure of calmodulin-dependent protein kinase I.

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Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.


Calcium/calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to the enzyme and indirectly promotes the phosphorylation and synergistic activation of CaMKI by an exogenous kinase. We have evaluated the initial CaM-dependent activation of the unphosphorylated form of CaMKI. The kinetics of bacterially expressed human CaMKI show that the peptide syntide-2 is a relatively poor substrate, whereas the synapsin site-1 peptide is 17-fold more specific. The peptide ADR1G is 400-fold more specific than syntide-2, and its catalytic rate is among the highest reported for a kinase peptide substrate. To understand how CaM activates CaMKI, we have characterized the activation of the enzyme by CaM mutants with substitutions at hydrophobic residues. The point mutant M124Q located in the C-terminal domain of CaM produced a 57-fold increase in the CaM activation constant for CaMKI and suggests the involvement of methionine 124 in an important hydrophobic interaction with tryptophan 303 of CaMKI. Substituting two, three, and five hydrophobic residues in the N-terminal domain of CaM increased the CaM activation constant for CaMKI by 10-190-fold and lowered the maximal enzyme activity by more than 80%. Two of these N-terminal mutants of CaM do not affect the Km for peptide substrate but instead produce a 5-10-fold higher Km for ATP. This result demonstrates the critical role of the N-terminal domain of CaM in regulating the access of ATP to CaMKI.

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