Heterogeneous Reaction of Heavy Meromyosin ATPase with 2′ (or 3′)-O-(2, 4, 6-Trinitrophenyl)Adenosine 5′-Triphosphate

Abstract

1. An initial burst of P₁ liberation (1.2 mol per mol of heavy meromyosin) was observed in the hydrolysis of 2′ (or 3′)-O-(2, 4, 6-trinitrophenyl)adenosine 5′-triphosphate(TNP-ATP) by heavy meromyosin ATPase [EC 3.6. 1.3]. When heavy meromyosin was preincubated with excess TNP-ATP, the size of the initial burst of P liberation from the added ATP increased with ATP concentration and reached a constant level (1.28 mol) at a 1.9 molar excess of ATP to the enzyme. 2. On preincubation of heavy meromyosin with excess TNP-ATP, the steady-state ATPase activity was reduced to below 20% of the control at low ATP concentrations (below 0.5 mM) without change in the size of the initial burst of P liberation. However, it recovered with increase of ATP concentration and reached the same level as the control at higher ATP concentrations (above 1 mM). The difference spectrum at 518 nm, which is induced by TNP-ADP interacting with a tryptophanyl residue of heavy meromyosin, decreased with increase of concentration of ATP added and almost disappeared at 0.9 m ATP, where the steady-state ATPase activity had recovered to 90% of the control. 3. On the reaction of heavy meromyosin with TNP-ATP, a complex of heavy meromyosin with 2 mol of TNP-ADP was isolated by dialysis. One mole of TNP-ADP was readily released on incubation with 0.2-0.4 mM ATP or by ammonium sulfate precipitation before dialysis. Higher concentrations of ATP (above 1 mM) were necessary to release the other 1 mol of TNP-ADP. 4. The results of the present study show differences between the two bindings of TNP-ADP to heavy meromyosin, one to the active site for the initial burst of P liberation and the other to that for the steady-state ATPase. The former is readily disrupted by adding low concentrations of ATP or by precipitation with ammonium sulfate, and the latter, involving interaction with a tryptophanyl residue, is stable even in the presence of sodium dodecyl sulfate.