Kinetic effects of kinesin switch I and switch II mutations.
We have examined several mutants in the switch I, switch II region of rat kinesin. Pre-steady-state kinetic analysis of association and dissociation of an N256K mutant with nucleotides and microtubules demonstrates that the mutation blocks microtubule stimulation of nucleotide release and ATP hydrolysis without affecting other kinetic parameters. The results suggest that ADP release on one head may be coupled to structural changes on the other head to stimulate ATP hydrolysis. Mutations at Glu(237), a residue predicted to participate in a hydrogen-bond interaction critical for nucleotide processing, reduced or abolished microtubule-dependent ATPase activity with only minor effects on pre-steady-state rates of nucleotide release or binding. Mutations at Glu(200), a residue that could serve as an alternate electron acceptor in the above-mentioned hydrogen-bond interaction, had small effects on microtubule-dependent ATPase activity despite modestly reducing the rate at which microtubule-stimulated nucleotide release occurs. These results further clarify the pathway of coupling of ATP hydrolysis to force production.
Auerbach
Scott D
SD
Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, USA.
Johnson
Kenneth A
KA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001921:Brain / Q000378:metabolism; D002417:Cattle; D019281:Dimerization; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006868:Hydrolysis; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000201:enzymology*; D009154:Mutation / Q000235:genetics*; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
D010710:Phosphates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases; D016547:Kinesins