Role of the pseudosubstrate sequence in smooth muscle myosin light chain kinase thermal stability

M. C. Faux, R. B. Pearson, K. I. Mitchelhill, B. E. Kemp

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5 Citations (Scopus)

Abstract

Smooth muscle myosin light chain kinase (MLCK) is stable in the presence of Ca2+/calmodulin and does not undergo inactivation as reported for 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/calmodulin binding sequence deleted undergoes rapid inactivation (t( 1/2 ) = 5 min at 25 °C). Thermal inactivation renders the 61-kDa fragment more susceptible to cleavage by trypsin. The pseudosubstrate sequence, smMLCK-(787-807) prevents inactivation with high potency (half-maximal protective concentration, PC0.5 = 102 ± 9 nM). The hexapeptide smMLCK-(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 to 500 nM. However, substitution of both aromatic residues Tyr794 and Trp800 in the longer pseudosubstrate peptide-(787-807) had little effect, indicating that with the longer peptide other multiple interactions were sufficient to stabilize the enzyme. The peptide substrate MLC-(11-23) A14,15 was also protective (PC0.5 = 380 nM) as was Mg2+-ATP, Mg2+-ADP, and Mg2+ plus adenosine. The results demonstrate that the sequence extending from 787-815 encoding the previously identified overlapping pseudosubstrate and calmodulin binding sequences also contains residues that are essential for maintaining thermal stability but these exhibit distinct structure/function relationships.

Original languageEnglish
Pages (from-to)12484-12491
Number of pages8
JournalThe Journal of Biological Chemistry
Volume268
Issue number17
Publication statusPublished - 1 Jan 1993
Externally publishedYes

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