The ATPase cycle of the mitotic motor CENP-E.

We have previously shown that the mitotic motor centrosome protein E (CENP-E) is capable of walking for more than 250 steps on its microtubule track without dissociating. We have examined the kinetics of this molecular motor to see if its enzymology explains this remarkable degree of processivity. We find that like the highly processive transport motor kinesin 1, the enzymatic cycle of CENP-E is characterized by rapid ATP binding, multiple enzymatic turnovers per diffusive encounter, and gating of nucleotide binding.

The mitotic kinesin CENP-E is a processive transport motor.

In vivo studies suggest that centromeric protein E (CENP-E), a kinesin-7 family member, plays a key role in the movement of chromosomes toward the metaphase plate during mitosis. How CENP-E accomplishes this crucial task, however, is not clear. Here we present single-molecule measurements of CENP-E that demonstrate that this motor moves processively toward the plus end of microtubules, with an average run length of 2.

Kinetics of structural changes in the relay loop and SH3 domain of myosin.

The intrinsic fluorescence of smooth muscle myosin signals conformational changes associated with different catalytic states of the ATPase cycle. To elucidate this relationship, we have examined the pre-steady-state kinetics of nucleotide binding, hydrolysis, and product release in motor domain-essential light chain mutants containing a single endogenous tryptophan, either residue 512 in the rigid relay loop or residue 29 adjacent to the SH3 domain. The intrinsic fluorescence of W512 is sensitive to both nucleotide binding and hydrolysis, and appears to report structural changes at the active site, presumably through a direct connection with switch II.

Nucleotide dependent intrinsic fluorescence changes of W29 and W36 in smooth muscle myosin.

The intrinsic fluorescence of smooth muscle myosin is sensitive to both nucleotide binding and hydrolysis. We have examined this relationship by making MDE mutants containing a single tryptophan residue at each of the seven positions found in the wild-type molecule. Previously, we have demonstrated that a conserved tryptophan residue (W512) is a major contributor to nucleotide-dependent changes of intrinsic fluorescence in smooth muscle myosin.