Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, P-K-K-A-K-R-R-A-A-E-G-S-S(P)-N-V-F-S where S(P) is phosphoserine, were K(m), 2.3 μM and V(max), 0.9 μmol/min/mg of enzyme. K(m) values were 122 and 162 μM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S(P)-N-V-F-S, were K(m), 1.4 μM and V(max), 27 μmol/min/mg of enzyme. Average K(m) values for the smooth muscle peptide, residues 11-23, were 10 μM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. V(max) values decreased and K(m) values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH₂ terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH₂ terminus from phosphoserin in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.