title: Biochemical characterization of the novel rice kinesin K23 and its kinetic study using fluorescence resonance energy transfer between an intrinsic tryptophan residue and a fluorescent ATP analogue.
abstract: We previously demonstrated that the rice kinesin K16, which belongs to the kinesin-7 subfamily, has unique enzymatic properties and atomic structure within key functional regions. In this study, we focused on a novel rice plant kinesin, K23, which also belongs to the kinesin-7 subfamily. The biochemical characterization of the K23 motor domain (K23MD) was studied and compared with the rice kinesin K16 and other related kinesins. K23 exhibits ∼45-fold (1.3 Pi mol(-1) site mol(-1) s(-1)) lower microtubule-dependent ATPase activity than conventional kinesins, whereas its affinity for microtubules is comparable with conventional kinesins. MgADP-free K23 is unstable compared with the unusually stable MgADP-free K16MD. The enzymatic properties of K23MD are somewhat different from those of K16. We used a fluorescent ATP analogue 2'(3')-O-(N'-methylanthraniloyl)-ATP (mant-ATP) for the kinetic characterization of K23. The fluorescence of mant-ATP was not significantly altered during its hydrolysis by K23. However, significant fluorescence resonance energy transfer (FRET) between mant-ATP and W21 in the motor domain was observed. The kinetic study using FRET revealed that K23 has unique kinetic characteristics when compared with other kinesins.
authors.affiliation: Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Umezu
authors.forename: Nobue
authors.initials: N
authors.lastname: Hanzawa
authors.forename: Masafumi D
authors.initials: MD
authors.lastname: Yamada
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kondo
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Mitsui
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Maruta
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D016547:Kinesins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D007527:Isoenzymes; D010940:Plant Proteins; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D014364:Tryptophan; D000255:Adenosine Triphosphate; D016547:Kinesins