KINETIC MECHANISM OF KINESIN MOTOR DOMAIN

The kinetic mechanism of the human kinesin ATPase motor domain K379, expressed in Escherichia coli, was determined by transient and steady-state kinetic studies. The steps in nucleotide binding were measured using the fluorescent substrate analogues, methylanthraniloyl ATP (mant-ATP) and mant-ADP. Both nucleotides gave a two-step fluorescence signal, an increase followed by a decrease, which indicates that two isomerizations are induced by nucleotide binding. The ATPase mechanism is fitted by a six-step reaction: K + T [GRAPHICS] K + D where, T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; K(T) and K(D) are states in rapid equilibrium with the free nucleotide. A set of kinetic constants for 20 degrees C 50 mM NaCl is K-1 = 2 x 10(4) M(-1), k(2) = 200 s(-1), k(3) = 9 s(-1), k(5) = 0.01 s(-1), and K-6 = 2 x 10(-5) M. Values of K-1 and K-6 are estimates for mant-ATP and mant-ADP, respectively. ADP dissociation is the rate-limiting step. The rate constant for a decrease in fluorescence for the transitions from the high fluorescence K . T state to the low fluorescence K . D state is equal to k(3), the rate constant of the hydrolysis step measured by quench flow experiments. The decrease could occur in step 3 or step 4 if k(4) > k(3). The binding of mant-ADP fitted the scheme K + D [GRAPHICS] K . D-2 where K-a = (1/K-6) = 5 x 10(4) M(-1); k(b) = 200 s(-1) is the rate constant of the transition to the high fluorscence K . D-1 state; k(c) = 40 s(-1) is the rate of the transition to the low fluorescence K . D-2 state (in 50 mM NaCl). The final level of enhancement is the same for mant-ADP and mant-ATP in the steady state, and the K . D-2, and K . D states have the same rate constant of mant ADP dissociation and are considered to be identical. The rate constants agreed closely with values obtained previously for the kinesin tetramer prepared from calf brain [Sadhu, A., & Taylor, E. W. (1992) J. Biol. Chem. 267, 11352-11359].