ROLE OF THE PSEUDOSUBSTRATE SEQUENCE IN SMOOTH-MUSCLE MYOSIN LIGHT-CHAIN KINASE THERMAL-STABILITY

Smooth muscle myosin light chain kinase (MLCK) is stable in the presen ce of Ca2+/calmodulin and does not undergo inactivation as reported fo r skeletal muscle MLCK (Kennelly, P. J., Starovasnik, M. A., Edelman, A. M., and Krebs, E. G. (1990) J. Biol. Chem. 265, 1742-1749). The 61- kDa tryptic fragment of smMLCK-(283-779) with the pseudosubstrate/calm odulin binding sequence deleted undergoes rapid inactivation (t1/2 = 5 min at 25-degrees-C). Thermal inactivation renders the 61-kDa fragmen t more susceptible to cleavage by trypsin. The pseudosubstrate sequenc e, smMLCK-(787-807) prevents inactivation with high potency (half-maxi mal protective concentration, PC0.5 = 102 +/- 9 nM). The hexapeptide s mMLCK-(797-802), Arg-Arg-Lys-Trp800-Gln-Lys, protected with a similar potency (PC0.5 = 73 +/- 14 nM). The four basic residues as well as Trp were important for maintaining protection by the hexapeptide smMLCK-( 797-802). Substitution of Trp800 with Ala or Leu increased the PC0.5 t o 500 nM. However, substitution of both aromatic residues Tyr794 and T rp800 in the longer pseudosubstrate peptide-(787-807) had little effec t, indicating that with the longer peptide other multiple interactions were sufficient to stabilize the enzyme. The peptide substrate MLC-(1 1-23) A14,15 was also protective (PC0.5 = 380 nM) as was Mg2+-ATP, Mg2 +-ADP, and Mg2+ plus adenosine. The results demonstrate that the seque nce extending from 787-815 encoding the previously identified overlapp ing pseudosubstrate and calmodulin binding sequences also contains res idues that are essential for maintaining thermal stability but these e xhibit distinct structure/function relationships.