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Mechanism of Inhibition of Skeletal Muscle Actomyosin by N -Benzyl- p -toluenesulfonamide

2003; American Chemical Society; Volume: 42; Issue: 20 Linguagem: Inglês

10.1021/bi026964f

1943-295X

Michael Shaw, E. Michael Ostap, Yale E. Goldman,

Muscle Physiology and Disorders

N-Benzyl-p-toluenesulfonamide (BTS) is a small organic molecule that specifically inhibits the contraction of fast skeletal muscle fibers. To determine the mechanism of inhibition by BTS, we performed a kinetic analysis of its effects on the elementary steps of the actomyosin subfragment-1 ATPase cycle. BTS decreases the steady-state acto-S1 ATPase rate approximately 10-fold and increases the actin concentration for half-maximal activation. BTS primarily affects three of the elementary steps of the reaction pathway. It decreases the rate of Pi release >20-fold in the absence of actin and >100-fold in the presence of actin. It decreases the rate of S1·ADP dissociation from 3.9 to 0.8 s-1 while decreasing the S1·ADP dissociation constant from 2.3 to 0.8 μM. BTS weakens the apparent affinity of S1·ADP for actin, increasing the Kd from 7.0 to 29.5 μM. ATP binding to S1, hydrolysis, and the affinity of nucleotide-free S1 for actin are unaffected by BTS. Kinetic modeling indicates that the binding of BTS to myosin depends on actin association/dissociation and on nucleotide state. Our results suggest that the reduction of the acto-S1 ATPase rate is due to the inhibition of Pi release, and the suppression of tension is due to inhibition of Pi release in conjunction with the decreased apparent affinity of S1·ADP·Pi and S1·ADP for actin.