CHARACTERIZATION OF SUBSTRATE PHOSPHORYLATION AND USE OF CALMODULIN MUTANTS TO ADDRESS IMPLICATIONS FROM THE ENZYME CRYSTAL-STRUCTURE OF CALMODULIN-DEPENDENT PROTEIN-KINASE-I

Calcium/calmodulin (CaM) directly activates CaM-dependent protein kina se I (CaMKI) by binding to the enzyme and indirectly promotes the phos phorylation and synergistic activation of CaMKI by an exogenous kinase . We have evaluated the initial CaM-dependent activation of the unphos phorylated 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 pept ide 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 activati on of the enzyme by CaM mutants with substitutions at hydrophobic resi dues. The point mutant M124Q located in the C-terminal domain of CaM p roduced a 57-fold increase in the CaM activation constant for CaMKI an d suggests the involvement of methionine 124 in an important hydrophob ic interaction with tryptophan 303 of CaMKI. Substituting two, three, and five hydrophobic residues in the N-terminal domain of CaM increase d 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 mut ants of CaM do not affect the K-m for peptide substrate but instead pr oduce a 5-10-fold higher K-m for ATP. This result demonstrates the cri tical role of the N-terminal domain of CaM in regulating the access of ATP to CaMKI.