Kinetic analysis of Dictyostelium discoideum myosin motor domains with glycine-to-alanine mutations in the reactive thiol region

Three conserved glycine residues in the reactive thiol region of Dictyostel ium discoideum myosin II were replaced by alanine residues. The resulting m utants G680A, G684A, and G691A were expressed in the soluble myosin head fr agment M761-2R [Anson, M., Geeves, M. A., Kurzawa, S. E., and Manstein, D. J. (1996) EMBO J. 15, 6069-6074] and characterized using transient kinetic methods. Mutant G691A showed no major alterations except for a marked incre ase in basal Mg2+-ATPase activity. Phosphate release seemed to be facilitat ed by this mutation, and the addition of actin to G691A stimulated ATP turn over not more than 3-fold. In comparison to M761-2R, mutant constructs G691 A and G684A showed a 4-fold reduction in the rate of the ATP cleavage step. Most other changes in the kinetic properties of G684A were small (similar to 2-fold). In contrast, substitution of G680 by an alanine residue led to large changes in nucleotide binding. Compared to M761-2R, rates of nucleoti de binding were 20-30-fold slower and the affinity for mantADP was approxim ately 10-fold increased due to a 200-fold reduction in the dissociation rat e constant of mantADP. The ATP-induced dissociation of actin from the acto. 680A complex was normal, but the communication between ADP and actin bindin g was altered such that the two sites are thermodynamically uncoupled but k inetically actin still accelerates ADP release.