Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide.
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 P(i) 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 microM. BTS weakens the apparent affinity of S1.ADP for actin, increasing the K(d) from 7.0 to 29.5 microM. 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 P(i) release, and the suppression of tension is due to inhibition of P(i) release in conjunction with the decreased apparent affinity of S1.ADP.P(i) and S1.ADP for actin.
Shaw
M Alexander
MA
Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia 19104, USA.
Ostap
E Michael
EM
Goldman
Yale E
YE
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D009119:Muscle Contraction / Q000187:drug effects; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D015879:Myosin Subfragments / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011721:Pyrenes / Q000737:chemistry; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013449:Sulfonamides / Q000494:pharmacology*; D014050:Toluene / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014364:Tryptophan / Q000737:chemistry
D000199:Actins; D004791:Enzyme Inhibitors; D015879:Myosin Subfragments; C444880:N-benzyl-p-toluene sulfonamide; D010710:Phosphates; D011721:Pyrenes; D013449:Sulfonamides; D014050:Toluene; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; C030984:pyrene