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, Lys11-Arg13, prevented phosphorylation. In contrast, deletion of Lys11-Arg13 did not completely abolish the phosphorylation for skeletal MLC kinase, and deletion of three additional residues was required for complete inhibition. Substitution of Arg16 with Glu markedly decreased Vmax for both smooth and skeletal MLC kinases. Substitution of Lys11-Arg13 with acidic or noncharged residues decreased Vmax, but these changes were much lower than that occurring on substitution of Arg16. Replacement of Lys11-Arg13 with acidic residues reduced the affinity of the free LC20 but had little effect on the myosin-incorporated LC20. 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 Arg16 but not the corresponding Glu13 of skeletal light chain resides in close proximity to Ser19, suggesting that the guanidyl group of Arg16 stabilizes the phosphate transfer and that the introduction of Glu at the 16th position would significantly destabilized this reaction.
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970.
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Ikebe
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Reardon
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Schwonek
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Sanders
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Ikebe
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005895:Gizzard, Avian; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014422:Turkeys
D017931:DNA Primers; D011994:Recombinant Proteins; D009219:Myosin-Light-Chain Kinase; D009218:Myosins