Kinetic characterization of the catalytic domain of Dictyostelium discoideum myosin.
The myosin head consists of a globular motor or catalytic domain that contains both the catalytic and actin binding sites, and a neck region which consists of a 8.5 nm alpha-helix that emerges from the globular part of the heavy chain and is stabilized by the binding of the essential and regulatory light chains. High levels of M754, a recombinant polyhistidine-tagged catalytic domain-like fragment of myosin II, were produced in Dictyostelium discoideum and purified using a rapid extraction protocol and metal chelate chromatography. Approximately 1.2 mg of homogeneous, functional protein was obtained per gram of cells. Kinetic analysis of M754 showed that the recombinant protein still has all the typical properties of a myosin ATPase. However, the removal of the light chain domain does have a pronounced effect on enzymatic activity. Nucleotide on-rates are 7-16-fold slower for M754 than for a myosin head fragment that includes the neck region. In contrast, the rate of ATP binding and dissociating the actin-bound catalytic domain is 10-fold increased. Overall the results indicate that the truncation of the heavy chain affects the nucleotide binding site and the communication between the nucleotide and actin binding sites. Furthermore, it seems that the nucleotide site of M754 is not fully formed but binding to actin or ATP stabilizes the structure in general and the nucleotide binding site in particular.
National Institute for Medical Research, London, U.K.
S K
SK
Woodward
M A
MA
Geeves
D J
DJ
Manstein
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004023:Dictyostelium / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016324:Sequence Tagged Sites
D017931:DNA Primers; D011994:Recombinant Proteins; D006639:Histidine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins