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 essen tial and regulatory light chains. High levels of M754, a recombinant p olyhistidine-tagged catalytic domain-like fragment of myosin II, were produced in Dictyostelium discoideum and purified using a rapid extrac tion protocol and metal chelate chromatography. Approximately 1.2 mg o f homogeneous, functional protein was obtained per gram of cells. Kine tic analysis of M754 showed that the recombinant protein still has all the typical properties of a myosin ATPase. However, the removal of th e light chain domain does have a pronounced effect on enzymatic activi ty. Nucleotide on-rates are 7-16-fold slower for M754 than for a myosi n head fragment that includes the neck region. In contrast, the rate o f ATP binding and dissociating the actin-bound catalytic domain is 10- fold increased. Overall the results indicate that the truncation of th e heavy chain affects the nucleotide binding site and the communicatio n between the nucleotide and actin binding sites. Furthermore, it seem s that the nucleotide site of M754 is not fully formed but binding to actin or ATP stabilizes the structure in general and the nucleotide bi nding site in particular.