INTERACTION OF MANT-ADENOSINE NUCLEOTIDES AND MAGNESIUM WITH KINESIN

Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluore scent transient. The pH and microtubule dependence of the rates and am plitudes indicates that the two phases are produced by release of boun d mant-ADP, with an excess of the 3'-isomer, followed by the subsequen t relaxation of the free 2'- and 3'-isomers to their equilibrium distr ibution. The first phase for release of mant-ADP is accelerated by mic rotubules and occurs at the same rate as ADP release measured using [P -32]ADP. The second phase is subject to base catalysis and occurs at t he same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomeriz ation rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomer s interconvert only slowly when bound to myosin. No fluorescence reson ance energy transfer occurs between the single tryptophan in the kines in neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in th e absence of microtubules is minimal (0.005 s(-1)) at pH 7-8, 2 mM Mg2 +, and 25 mM KCl but is accelerated at lower pH (0.04 s(-1) at pH 5.5) and either lower or higher [KCl] (0.01 and 0.06 s(-1) at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monoval ent salts. Reduction of the free Mg2+ by addition of excess EDTA incre ases the release of mant-ADP from E.MgADP to 0.03 s(-1). This accelera tion at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s(-1) followed by rapid release of ADP, as the rate of ADP release fro m Mg-free E.ADP is fast (>0.5 s(-1)). At high Mg2+, rebinding of Mg2to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s(-1) .