Elucidating the molecular mechanisms by which calcium/calmodulin activated enzymes aid transformation of regulatory calcium-signals into biochemical responses is the ultimate goal. The proposal focuses on type II calmodulin- dependent protein kinase, a dodecameric enzyme of functionally identical subunits; each subunit is composed of catalytic, regulatory, and association domains. As the location of the ATP-binding site has merely been inferred by sequence comparison with related protein kinases, and the corresponding site for protein substrates is unknown, these continuing investigations are directed towards characterizing both of these substrate-binding sites. Because ligand-promoted instability and aggregation have hampered studies with the holoenzyme, a catalytic fragment will be used. This monomeric domain has the added advantage of needing neither calmodulin nor autophosphorylation to be fully active. This domain will be characterized and used to probe the kinetic reaction mechanism, to study substrate-binding, and to identify amino acid side chains which are lining the two substrate-binding pockets. The location of the ATP-binding site on the protein sequence of the kinase will be verified by affinity and photoaffinity labeling with analogs of ATP. Photoaffinity labeling with photoreactive peptide substrates will be used to locate the second site. Calcium is one of the most important intracellular regulators and second messengers of biological systems. The intracellular levels of Ca2+ are increased upon stimulation by a variety of extracellular agents, such as hormones, drugs, and neurotransmitters. The increase in the concentration of Ca2+ triggers a serious of regulatory events that ultimately alter many processes, including those responsible for muscle contraction and the transmission of nerve impulses. Activation of calmodulin-dependent protein kinase and calcinerin seem to be part of the mechanism by which the Ca2+ calmodulin complex initiates biochemical responses at the synapse because a detailed understanding of the structure, function and regulation of these enzymes may aid in elucidating the molecular events at the synapse, studies of these interconverting enzymes are of considerable interest.
