Nucleotide-free kinesin hydrolyzes ATP with burst kinetics.
Bovine brain kinesin binds ADP tightly and contains a stoichiometric amount of ADP at its active site when isolated in the presence of free Mg2+ (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318). EDTA in excess of Mg2+ weakens ADP binding and nucleotide-free kinesin can be prepared by gel filtration with excess EDTA. On addition of ATP, this nucleotide-free enzyme catalyzes the rapid hydrolysis of a stoichiometric amount of ATP in a burst phase followed by much slower continued ATP hydrolysis limited by the release of ADP from the active site. This burst reaction is evident both by formation of [32P]Pi from [gamma-32P]ATP and by formation of [alpha-32P]ADP from [alpha-32P]ATP. At 1.1 nM kinesin active sites, the observed rate of the burst phase increases linearly with ATP over the 1-20 nM range yielding a bimolecular rate of net ATP binding and hydrolysis of 2.5 microM-1 s-1. The intercept at zero ATP is 0.008 s-1 which equals the ADP release rate at 0.008-0.009 s-1. This predicts a Km for ATP of approximately 3.5 nM and measurements of the dependence on ATP concentration of the steady state rate and amount of bound ADP are consistent with a Km of this magnitude.
Hackney
D D
DD
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Malik
A S
AS
Wright
K W
KW
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D006868:Hydrolysis; D016547:Kinesins; D007700:Kinetics; D008868:Microtubule Proteins / Q000378:metabolism
D008868:Microtubule Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins