Kinetic and spectroscopic evidence for three actomyosin : ADP states in smooth muscle

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin I I. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to a n identifiable state change. These studies indicate that for this myosin:AD P, both the catalytic site and the actin-binding site can each assume one o f two conformations. Relatively loose coupling between these two binding si tes leads to three discrete actin-associated ADP states, Following an initi al, weakly bound state, binding of myosin: ADP to actin shifts the equilibr ium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, i ncluding Dictyostelium myosin II, have reciprocal coupling between the acti n- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly b ound actomyosin ADP state in smooth muscle, would prolong the fraction of t he ATPase cycle time that this actomyosin spends in a force-generating conf ormation and may be central to explaining the physiologic differences betwe en this and other myosins.