Spatial requirements for location of basic residues in peptide substrates for smooth muscle myosin light chain kinase.
The requirement of basic residues as substrate specificity determinants for the chicken gizzard myosin light chain kinase has been studied using synthetic peptide analogs of the local phosphorylation site sequence in the myosin light chains, Lys-Lys-Arg13-Pro-Gln-Arg16-Ala-Thr-Ser19-Asn-Val-Phe- Ala. The basic residue, Arg-16, was found to have a strong influence on the kinetics of phosphorylation similar to that reported previously for the three adjacent residues, Lys-11, Lys-12, and Lys-13 (Kemp, B. E., Pearson, R. B., and House, C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7471-7475). The location of Arg-16 in relation to Ser-19 as well as the distance between Arg-13 and Arg-16 had a profound effect on both the kinetics and the site specificity of phosphorylation. Placement of Arg-16 at position 15 resulted in a complete switch in phosphorylation site specificity from Ser-19 to Thr-18. Increasing the number of alanine residues between Arg-13 and Arg-16 in the model peptide, Lys-Lys-Arg-(Ala)n-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala, also influenced the kinetics and site specificity of peptide phosphorylation. With two or three alanines (n = 2 or 3), the apparent Km was 7.5 and 10 microM, respectively, and 97% of the phosphate was esterified to Ser-19. Increasing or decreasing the number of alanines (n = O to n = 4) was accompanied by an increase in the apparent Km and phosphorylation of both Thr-18 and Ser-19. These results support the concept that both the presence and location of basic residues play an essential role in the substrate specificity of the smooth muscle myosin light chain kinase.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013329:Structure-Activity Relationship
D010455:Peptides; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase