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Abstract

We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp−501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501 Y mutant in the soluble myosin head fragment M761−2R (W501Y−2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6−fold decreased, but other kinetic properties of the protein were changed less than 2−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−sensitive 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 emission