Observation of multiple myosin subfragment 1-ADP-fluoroberyllate complexes by 19F NMR spectroscopy.
Fluoroaluminate and fluoroberyllate are potent inhibitors of the ATPase activity of myosin. Inhibition requires the presence of ADP, and much evidence has accumulated to suggest that the tetrahedral fluoroaluminate and fluoroberyllate ions act as phosphate analogues, binding with high affinity at the active site in the position normally occupied by the terminal phosphate of ATP. Both the S1-ADP-fluoroaluminate and the S1-ADP-fluoroberyllate species are thought to resemble kinetic intermediates in the actomyosin ATPase cycle. Characterization of S1-bound fluoroaluminate by 19F NMR is straightforward; a single resonance identified as AlF4- is observed easily [Maruta, S., Henry, G.D., Sykes, B.D., & Ikebe, M (1993) J. Biol. Chem. 268, 7093-7100]. Bound fluoroberyllate, by contrast, was found to give rise to four separate peaks: a downfield pair at -80 and -83.5 ppm and an upfield pair at -101.5 and -103 ppm, suggesting the existence of four distinct types of S1-ADP-fluoroberyllate complex. The relative intensities of the bound resonances can be altered by changing rhe F:Be ratio during complex formation. Integration of a spectrum acquired in the presence of a fluorine-labeled nucleotide derivative, 3'(2')-O-(4-fluorobenzoyl)-ADP, in place of ADP yielded a bound fluoride to nucleotide ratio of 1.7-1.9 to 1, showing that the major bound fluoroberyllate species cannot be BeF3- as is usually thought. It is proposed that the bound fluoroberyllates correspond to the neutral species BeF2(H2O)2 and BeFOH(H2O)2 and the negatively charged species [BeF2OH.H2O]- and [BeF3.H2O]-, although other possibilities are discussed.
Department of Biochemistry, University of Alberta, Edmonton, Canada.
G D
GD
Henry
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S
Maruta
M
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Ikebe
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Sykes
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D001608:Beryllium / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000037:antagonists & inhibitors*; D011487:Protein Conformation; D012996:Solutions
D015879:Myosin Subfragments; D012996:Solutions; D000244:Adenosine Diphosphate; D009218:Myosins; D001608:Beryllium; D005459:Fluorides