Abstract

Numerous analytical experiments have shown that, in solution, ATP analogues with bulky substitutions at the eighth position of the adenine ring predominantly assume the syn conformation with respect to the adenine-ribose bond. Two such analogues, 3'-O-(N-methylanthraniloyl)-8-azido-ATP (Mant-8-N3-ATP) and 8-Br-ATP, were synthesized and used to probe the conformation of the ATP-binding site of myosin. In the presence of these analogues, actomyosin was rapidly dissociated; Mg2+-dependent ATP hydrolysis was significantly activated by actin; and Pi bursting was observed. For skeletal myosin, however, these analogues failed to support actin translocation, and they did not significantly enhance the intrinsic tryptophan fluorescence of skeletal muscle myosin subfragment-1 (SKE S-1). These results suggest that although myosin**/ADP/Pi intermediates can be formed with these analogues, the crucial conformational changes required for cross-bridge cycling do not occur in skeletal muscle myosin. The conformations of the ATP-binding sites of skeletal and smooth-muscle myosin were compared using the ternary complexes, myosin-ADP-beryllium fluoride (BeFn) or myosin-ADP-aluminium fluoride (AIF4-). In AlF4- complexes, Mant-8-N3-ADP affinity labeled the N-terminal 29-kDa domain of smooth-muscle myosin subfragment-1 (SM S-1), as did ATP analogues having the anti conformation, whereas it labeled the C-terminal 20-kDa domain of skeletal S-1. In smooth muscle BeFn complexes, Mant-8-N3-ADP was equally likely to cross-link to the 29-kDa N-terminal and the 25-kDa C-terminal domains. These analogues induced smooth muscle actomyosin super-precipitation and increased intrinsic tryptophan fluorescence to the same degree as ATP itself. As was expected from above results, the analogues supported smooth-muscle-myosin-induced actin translocation. These results suggest that smooth-muscle myosin adopts the eight-substituted ATP analogue in the normal conformation, but skeletal muscle myosin does not. This reflects the likely differences in the structures of their respective ATPase sites.