Role of highly conserved lysine 130 of myosin motor domain. In vivo and in vitro characterization of site specifically mutated myosin.
We have created a mutant Dictyostelium myosin II heavy chain gene in which a highly conserved lysine residue (Lys-130) is changed to leucine. Lys-130 is a residue that is known to be trimethylated in skeletal muscle myosin and had been thought to play an integral role in the interaction of myosin with ATP during the actomyosin chemomechanical cycle. We report here the first in vivo and in vitro characterization of an engineered missense mutation in the motor domain of myosin. Expression of the K130L myosin in a Dictyostelium strain that lacks the myosin II heavy chain gene is sufficient to restore the ability of that cell line to undergo cytokinesis and multicellular development, processes that require functional myosin. The K130L myosin purified from these cells displays maximal actin-activated ATPase activities and promotes maximal sliding velocities of actin filaments in an in vitro motility assay that are comparable with those of wild-type myosin. These results demonstrate that this lysine residue is not required for the enzymatic or motile activities of myosin. However, the mutant protein exhibits a 4-fold increase in Km for ATP over wild-type myosin, indicating that this residue participates in the interaction of myosin with its nucleotide substrate.
Ruppel
K M
KM
Department of Biochemistry, Stanford University School of Medicine, California 94305.
Uyeda
T Q
TQ
Spudich
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002465:Cell Movement; D004023:Dictyostelium / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D009218:Myosins; D008239:Lysine