title: Mutants of smooth muscle myosin light chain kinase at tryptophan 800.
abstract: The importance of Trp 800 in the calmodulin-binding site of myosin light chain kinase was investigated. Truncation mutants from Leu 447 to the C-terminus were expressed in E. coli and these were modified by point mutations of Trp 800 to Gly, Cys, Leu and Tyr. Trp at this position was more effective than any of the other residues. The Leu mutant was partially active and its Km for calmodulin decreased from about 10 nM to 175 nM. The Tyr mutant had detectable activity but the other two mutants were inactive and did not bind calmodulin. Thus Trp at position 800 is critical. The activity of the Leu mutant at high calmodulin concentrations was less than the wild-type mutant, about 20%. This suggests that the binding of calmodulin does not release inhibition in an all-or-none mechanism and that other intramolecular interactions are important.
authors.affiliation: Muscle Biology Group, University of Arizona, Tucson 85721.
authors.forename: S
authors.initials: S
authors.lastname: Matsushima
authors.forename: Y P
authors.initials: YP
authors.lastname: Huang
authors.forename: C V
authors.initials: CV
authors.lastname: Dudas
authors.forename: V
authors.initials: V
authors.lastname: Guerriero
authors.forename: D J
authors.initials: DJ
authors.lastname: Hartshorne
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006041:Goats; D009130:Muscle, Smooth / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D014364:Tryptophan / Q000737:chemistry*; D014422:Turkeys
chemical_list: D002147:Calmodulin; D014364:Tryptophan; D009219:Myosin-Light-Chain Kinase; D002118:Calcium