Nucleotide-induced closure of the ATP-binding pocket in myosin subfragment-1.
The hydrophobic fluorescent dye 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phen ylboronic acid (PPBA) stoichiometrically binds to myosin subfragment-1 (S-1) with a dissociation constant (Kd) of 26 microns. Upon addition of nucleotides to the complex of S-1 with PPBA, the PPBA-binding site is converted to a more firm hydrophobic pocket accompanied by a large fluorescence change. For the complex formed with ATP, binding of the dye is competitive with respect to ATP with a Ki of 0.8 microns. This competition was confirmed by the result that the Kd value of S-1 with ATP was increased 3.3-fold by the addition of PPBA. The dye bound to S-1 senses ATP hydrolysis but has no effect on the binding of actin to S-1. Measurements of acrylamide fluorescence quenching of the complexes have revealed that the Stern-Volmer quenching constant of the dye is reduced by a factor of 2.7 during ATP hydrolysis. These results together with the fluorescent properties of the complexes suggest that the ATP-binding pocket of S-1 is closed and/or tightened during ATP hydrolysis.
Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
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Hiratsuka
D000227:Adenine Nucleotides / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D009218:Myosins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000227:Adenine Nucleotides; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D009218:Myosins