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Inhibition of myosin ATPase by beryllium fluoride
Abstract:
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.
Insights
Beryllium fluoride inhibits myosin ATPase by binding to the M.ADP state, forming a stable complex through a multi-step process. This inhibition mechanism is similar to that observed with vanadate.
Area of Science:
- Biochemistry
- Enzymology
- Muscle Physiology
Background:
- Myosin subfragment 1 (S-1) is a key motor protein involved in muscle contraction.
- Understanding the regulation of myosin ATPase activity is crucial for elucidating muscle function.
- Beryllium fluoride (BeF3-) is known to mimic phosphate transition states in enzyme inhibition.
Purpose of the Study:
- To investigate the mechanism by which beryllium fluoride inhibits myosin S-1 ATPase activity.
- To determine the binding state and kinetic pathway of beryllium fluoride inhibition.
- To compare the inhibitory effects of beryllium fluoride with vanadate.
Main Methods:
- Studying the inhibition of myosin S-1 ATPase activity in the presence of MgATP and MgADP.
- Analyzing the kinetics of inhibition, including rates and dependence on BeF3- concentration.
- Utilizing tryptophan fluorescence and epsilon ADP chase experiments to characterize the inhibited complex.
Main Results:
- Beryllium fluoride inhibits myosin ATPase slowly, with rates analogous to vanadate inhibition.
- Inhibition kinetics suggest BeF3- binds to the M.ADP state of myosin.
- A multi-step inhibition process was identified, involving rapid binding, slow isomerization, and irreversible complex formation.
- The M++.ADP.BeF3- complex exhibits altered fluorescence properties and reduced epsilon ADP quenching.
Conclusions:
- Beryllium fluoride acts as an inhibitor of myosin S-1 ATPase by binding to the M.ADP state.
- The inhibition pathway involves a stable M++.ADP.BeF3- complex, analogous to M++.ADP.Vi and M**.ADP.Pi states.
- These findings provide insights into the transition state analog inhibition of myosin ATPase.