Movement of a kinesin heterodimer with one dead head

The eukaryotic motor enzyme kinesin performs work in the cell by transporting various cargoes along microtubules, and has the ability to do so as an isolated enzyme. Kinesin moves in discrete 8 nm steps with each step tightly coupled to the hydrolysis of 1 ATP molecule. Kinesin is functional in this capacity only as a dimer of identical motor, or "head" domains that are connected by a long helical coiled-coil. In vitro motility of kinesin is highly processive and unidirectional, even against substantial opposing load. Such high duty ratio motility is accounted for in "alternating-catalysis hand-over-hand" models, which features the two heads undergoing mandatory alternation between catalytic and non-catalytic states with each mechanochemical step. It is often hypothesized that such alternation in catalysis is essential to enable high duty ratio motility for kinesin. I created a method to independently modify the genetics of each head domain by co-expressing separate forms of kinesin in the same cell, thus allowing them to independently form kinesin heterodimers. I tested whether kinesin requires alternation in catalysis by pairing a mutant subunit (R210K) that is defective in ATP hydrolysis with a wild-type subunit. The resulting heterodimer was a functional and robust motor, and moved with a high duty ratio, like the wild-type motor. The heterodimer displayed evidence of alternate kinetics under low ATP/low opposing force conditions, but such evidence was undetectable under saturating ATP/high opposing force conditions. The lifetime of nucleotides resident on the heterodimer were measured. In contrast to the wild-type enzyme, the nucleotide residence lifetimes were independent of ATP concentration and therefore inconsistent with alternating catalysis models. I propose a single-site catalysis model that rationalizes the motility of the heterodimer under different conditions with the use of only one catalytic head.