Enhanced stimulation of myosin subfragment 1 ATPase activity by addition of negatively charged residues to the yeast actin NH2 terminus.
We examined the effects of yeast actin NH2-terminal mutations on actomyosin interactions and the function of actin in vivo through measurements of actin-activated ATPase activity, cosedimentation with rabbit muscle myosin subfragment 1 (S-1), in vitro motility, and invertase secretion assays. As reported earlier (Cook, R. K., Blake, W., and Rubenstein, P. A. (1992) J. Biol. Chem. 267, 9430-9436), elimination of NH2-terminal acidic residues from yeast actin results in an increased actin bundling, decreased actin-activated S-1 ATPase, and complete inhibition of actin filament sliding over myosin. Here we show that the addition of 2 new acidic residues to the NH2 terminus of yeast actin increased the Vmax value and the catalytic efficiency of the actin-activated ATPase activity of S-1. However, the binding of actin to S-1 in the presence of ATP and the velocities of actin sliding over myosin in the in vitro motility assays were not affected by this mutation. Thus, the number of actin NH2-terminal negative charges is important for actin activation of myosin S-1 ATPase activity, while only a minimum number of acidic residues is required for actin sliding over myosin in vitro. The number of actin NH2-terminal negative charges therefore appears to determine the efficiency with which the energy from ATP hydrolysis is converted to filament sliding.
Cook
R K
RK
Department of Biochemistry, College of Medicine, University of Iowa, Iowa City 52242.
Root
D
D
Miller
C
C
Reisler
E
E
Rubenstein
P A
PA
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013329:Structure-Activity Relationship; D043324:beta-Fructofuranosidase
D000199:Actins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D009838:Oligodeoxyribonucleotides; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D009218:Myosins