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 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 (t1/2 = 5 min at 25 degrees 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 Mg(2+)-ATP, Mg(2+)-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.
St. Vincent's Institute of Medical Research, Fitzroy Victoria, Australia.
M C
MC
Faux
R B
RB
Pearson
K I
KI
Mitchelhill
B E
BE
Kemp
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics; D013997:Time Factors; D014357:Trypsin
D002147:Calmodulin; D010446:Peptide Fragments; D010455:Peptides; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin