Specificity and kinetic effects of nitrophenol analogues that activate myosin subfragment 1

Abstract

2,4-Dinitrophenol (DNP) activates the myosin ATPase of mammalian skeletal muscle in the presence of Ca2+ or Mg2+, and inhibits it when the bivalent cations are replaced by K+ and EDTA. Activation of Mg2+ATPase is abolished by the presence of unregulated actin. 3-Nitrophenol (3-NP) is also an activator, whereas other analogues (2-nitrophenol, 2-NP, and 4-nitrophenol, 4-NP) are much less effective. Concentrations required for their half-maximal effects (K0.5) range from 2 to 15 mM for 3-NP and DNP in the presence of different cations, and the sequence for the analogues is 3-NP ⩽ DNP ≪ 2-NP ≈ 4-NP, which is apparently unrelated to either hydrophobicity or pK. DNP and 3-NP have almost identical effects on the ATPase activity of chymotryptic subfragment 1 as they do on myosin, which is an indication that their target is the globular head region rather than the tail, or the 18 kDa (regulatory) light chain. Analysis of the ATP concentration dependence for subfragment-1 ATPase in the presence of Ca2+ or Mg2+ shows that DNP activates only at high substrate concentrations, becoming increasingly effective with ATP concentrations in the physiological range. At low substrate concentrations, DNP inhibits hydrolysis by increasing the apparent Km for ATP at the catalytic site. In the presence of Mg2+, it mimics the effect of actin, which increases the Km and accelerates the release of products following hydrolysis. At high substrate concentrations, activation by DNP appears to involve a kinetic component with low affinity for ATP that can increase the overall reaction rate by a factor of 2- to 9-fold, depending on the bivalent cation. This low-affinity component is either induced by the drug (in the presence of Mg2+) or shifted by the drug to a lower ATP concentration range (in the presence of Ca2+).