Inhibition of myosin ATPase by beryllium fluoride.
Inhibition of the myosin subfragment 1 (S-1) ATPase activity by beryllium fluoride was studied directly in the presence of MgATP and following preincubation of samples with MgADP. In both cases, the rates of inhibition were very slow, with kapp = 0.5 and 58 M-1 s-1, respectively, in analogy to the rates of inhibition of myosin ATPase by vanadate [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2620-2624]. The very different rates of inhibition in the presence of MgATP and on preincubation with MgADP suggested that beryllium fluoride binds to the M.ADP state of myosin. The slow inhibition rates and the nonlinear dependence of the observed rates on beryllium fluoride concentration were consistent with a two-step inhibition process involving a rapid binding equilibrium to yield a collisional complex, M.ADP.BeF3-, and its slow isomerization into M++.ADP.BeF3-. A third, much slower, step was required to account for the conversion of the stable M++.ADP.BeF3- to a virtually irreversibly inhibited complex. Kinetic description of the inhibition pathway was derived from the observed rates of inhibition of myosin ATPase, information on the binding of beryllium fluoride to M.ADP, and measurements of epsilon ADP chase from M++.epsilon ADP.BeF3-. The isomerization rate and equilibrium constants were 1.4 x 10(-2) s-1 and 50, respectively, and the overall binding constant of beryllium fluoride to M.ADP was 5 x 10(5) M-1. The inhibitory complex showed a 16% enhancement to tryptophan fluorescence of S-1 and a reduced quenching of epsilon ADP by acrylamide. It is concluded that M++.ADP.BeF3- is analogous to the M++.ADP.Vi and M**.ADP.Pi states of myosin.
Phan
B
B
Department of Chemistry, University of California, Los Angeles 90024.
Reisler
E
E
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001608:Beryllium / Q000494:pharmacology*; D001665:Binding Sites; D004795:Enzyme Stability / Q000187:drug effects; D005459:Fluorides; D046911:Macromolecular Substances; D015879:Myosin Subfragments / Q000737:chemistry; D009218:Myosins / Q000037:antagonists & inhibitors*; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D015879:Myosin Subfragments; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D009218:Myosins; D001608:Beryllium; D005459:Fluorides