Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket.
A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin has been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myosin filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To test this hypothesis, loops derived from different myosin II isoforms that display a range of actin filament sliding velocities were inserted into a smooth muscle myosin backbone. Chimeric myosins were produced by baculovirus/Sf9 cell expression. Although the nature of this loop affected the rate of ADP release (up to 9-fold), in vitro motility (2.7-fold), and the Vmax of actin-activated ATPase activity (up to 2-fold), the properties of each chimera did not correlate with the relative speed of the myosin from which the loop was derived. Rather, the rate of ADP release was a function of loop size/flexibility with the larger loops giving faster rates of ADP release. The rate of actin filament translocation was altered by the rate of ADP release, but was not solely determined by it. Through a combination of solute quenching and transient fluorescence measurements, it is concluded that, as the loop gets smaller, access to the nucleotide pocket is more restricted, ATP binding becomes less favored, and ADP binding becomes more favored. In addition, the rate of ATP hydrolysis is slowed.
Sweeney
H L
HL
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. lsweeney@mail.med.upenn.edu
Rosenfeld
S S
SS
Brown
F
F
Faust
L
L
Smith
J
J
Xing
J
J
Stein
L A
LA
Sellers
J R
JR
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009068:Movement / Q000502:physiology*; D018482:Muscle, Skeletal; D009130:Muscle, Smooth; D009206:Myocardium; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity
D000199:Actins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins