Functional characterisation of Dictyostelium myosin II with conserved tryptophanyl residue 501 mutated to tyrosine

We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant my osin alone, when expressed in a Dictyosteliom strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular develo pment, processes which require a functional myosin in Dictyostelium, Additi onally, we expressed the W501Y mutant in the soluble myosin head fragment M 761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myos in motor domain. The affinity of the mutant myosin for actin was approximat ely 6-fold decreased, but other kinetic properties of the protein were chan ged less than e-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensi tive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emi ssion.