Equilibrium and rapid kinetic studies of the effect of N-ethylmaleimide on the binding of ADP to myosin, and H-meromyosin

Reaction of myosin or H-meromyosin with 10–20 moles of N-ethylmaleimide (NEM) per mole of enzyme inhibits the Mg++, Ca++ or EDTA-moderated ATPase activity, while the affinity of the enzyme for ADP increases. Inhibition of ADP binding requires 40–200 moles of NEM per mole of enzyme, suggesting the involvement of distinct classes of SH groups in the catalytic function and in the binding of ADP to myosin. Rapid kinetic measurements in a stopped-flow apparatus indicate that small concentrations of N-ethylmaleimide (2–5 moles per mole of enzyme) which activates the Mg2+ or Ca2+-moderated ATPase activity at 25 °, and inhibits at 6 °, decrease the rate of appearance and the magnitude of the ATP- or ADP-induced difference spectrum of H-meromyosin at 288 mμ. Assuming that the rate of appearance of the difference spectrum is a measure of the rate of interaction of ADP with H-meromyosin, SH group modification decreases both the formation (k1) and dissociation (k2) rate constants of the H-meromyosin-ADP complex. The decrease in k2 is accompanied by the inhibition of Mg-moderated ATPase activity by N-ethylmaleimide at 6 °. The ultraviolet difference spectrum at 288 mμ, which accompanies the binding of ATP or ADP to H-meromyosin is abolished by N-ethylmaleimide without major interference with the substrate binding.