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J. Biol. Chem., Vol. 269, Issue 44, 27251-27257, Nov, 1994

Nucleotide-induced closure of the ATP-binding pocket in myosin subfragment-1

T Hiratsuka
Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.

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.
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