Mechanoenzymes under superstall and large assisting loads reveal structural features.

Single-molecule experiments on the motor protein kinesin have observed runs of backsteps and thus a negative, that is, reverse mean velocity, V, under superstall loads, F; but, counterintuitively, beyond stall, V(F) displays a shallow minimum and then decreases in magnitude. Conversely, under assisting loads V(F) rises to a maximum before decreasing monotonically. By contrast, while the velocity of myosin V also saturates under assisting loads, the motor moves backward increasingly rapidly under superstall loads. For both kinesin and myosin V this behavior is implied remarkably well by simple two-state kinetic models when extrapolated to large loads. To understand the origins of such results in general mechanoenzymes, biochemical kinetic descriptions are discussed on the basis of a free-energy landscape picture. It transpires that the large-load performance is determined by the geometrical placement of the intermediate mechanochemical states of the enzymatic cycles relative to the associated transition states. Explicit criteria are presented for N-state sequential kinetics, including side-reaction chains, etc., and for parallel-pathway models. Physical colocalization of biochemically distinct states generally implies large-load velocity saturation.