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 Dictyostelium discoideummyosin II were replaced by alanine residues. The resulting mutants G680A, G684A, and G691A were expressed in the soluble myosin head fragment 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 increase in basal Mg2+-ATPase activity. Phosphate release seemed to be facilitated by this mutation, and the addition of actin to G691A stimulated ATP turnover not more than 3-fold. In comparison to M761-2R, mutant constructs G691A 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 ( approximately 2-fold). In contrast, substitution of G680 by an alanine residue led to large changes in nucleotide binding. Compared to M761-2R, rates of nucleotide binding were 20-30-fold slower and the affinity for mantADP was approximately 10-fold increased due to a 200-fold reduction in the dissociation rate constant of mantADP. The ATP-induced dissociation of actin from the acto.680A complex was normal, but the communication between ADP and actin binding was altered such that the two sites are thermodynamically uncoupled but kinetically actin still accelerates ADP release.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000818:Animals; D004023:Dictyostelium / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry*