PHOTOAFFINITY-LABELING OF A PEPTIDE SUBSTRATE TO MYOSIN LIGHT-CHAIN KINASE

The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the phot oreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino-t erminal of the phosphoacceptor serine (BpaKKRAAR-ATSNVFA), When photol yzed at 350 nm, the peptide was cross-linked stoichiometrically to myo sin light chain kinase in a Ca2+/calmodulin-dependent manner, Peptide incorporation into kinase inhibited light chain phosphorylation, and t he loss of kinase activity was proportional to the extent of peptide i ncorporated, After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodu lin, The extent of phosphorylation increased in the presence of Ca2+/c almodulin.The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and ana lysis, The covalent modification in the catalytic core occurred on Ile -373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14 %), respectively, Lys-572 in the autoinhibitory region accounted for 2 0% of the incorporated label, The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalyt ic site, Based upon a model of the catalytic core, the substrate pepti de is predicted to bind in the cleft between the two lobes of the kina se, The orientation of the substrate peptide on myosin light chain kin ase is similar to the orientation of the substrate recognition fragmen t, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-de pendent protein kinase.