STRUCTURAL REQUIREMENT OF THE REGULATORY LIGHT-CHAIN OF SMOOTH-MUSCLE MYOSIN AS A SUBSTRATE FOR MYOSIN LIGHT-CHAIN KINASE

The substrate structure required for skeletal and smooth muscle myosin light chain kinases (MLC kinase) was studied by using various mutant regulatory light chains of smooth muscle myosin. The deletion of the NH2-terminal 10 residues did not greatly affect the kinetic parameters of smooth MLC kinase; however, deletion of an additional 3 residues, Lys(11)-Arg(13), prevented phosphorylation. In contrast, deletion of Lys(11)-Arg(13) did not completely abolish the phosphorylation for skeletal MLC kinase, and deletion of three additional residues was required for complete inhibition. Substitution of Arg(16) with Glu markedly decreased V-max for both smooth and skeletal MLC kinases. Substitution of Lys(11)-Arg(13) with acidic or noncharged residues decreased V-max, but these changes were much lower than that occurring on substitution of Arg(16). Replacement of Lys(11)-Arg(13) with acidic residues reduced the affinity of the free LC(20) but had little effect on the myosin-incorporated LC(20). These results were different from those previously obtained with synthetic peptide analogs (Kemp, B. E., Pearson, R. B., and House, C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7471-7475) and suggest that a cluster of the basic amino acid residues are not fundamentally important for substrate recognition. The structural simulation revealed that the guanidyl group of Arg(16) but not the corresponding Glu(13) of skeletal Light chain resides in close proximity to Ser(19), suggesting that the guanidyl group of Arg(16) stabilizes the phosphate transfer and that the introduction of Glu at the 16th position would significantly destabilize this reaction.