Role of basic residues in the phosphorylation of synthetic peptides by myosin light chain kinase.

Abstract

The substrate specificity of the chicken gizzard myosin light chain kinase has been studied by using a series of synthetic peptide analogs of the NH2-terminal sequence of the chicken gizzard myosin light chain (Mr = 20,000). An 18-residue synthetic peptide, (sequence in text) corresponding to the sequence reported by Maita et al. [Maita, T., Chen, J. I. & Matsuda, G. (1981) Eur. J. Biochem. 117, 417-424], was phosphorylated with a 22-fold higher Km and a Vmax that was decreased to 1% of the native protein substrate. This peptide was also an inferior substrate when compared with an 18-residue synthetic peptide with an alternative sequence, (sequence: see text) which was phosphorylated with an apparent Km of 6.9 microM, comparable to the native protein substrate of 8.6 microM, and a Vmax of 3.9 mumol X min-1 X mg-1, 11% of that for the protein substrate. The kinetic of phosphorylation of shortened peptides corresponding to both sequences, together with peptides with appropriate substitutions, indicated that basic residues were the primary determinants of specificity for the smooth muscle myosin light chain kinase. In the latter peptide sequence, lysine residues 11 and 12 and the arginine at position 13 had a major influence on the kinetics of peptide phosphorylation.