Figure 4

Mutagenesis of the monastrol binding site in human Eg5. (A) Design of mutations in the human Eg5 motor domain. Amino acid side chains that are not conserved between Eg5 and conventional kinesin were targeted, either introducing chimeric or rationally designed mutations (asterix for targeted point mutants) to test the contribution of residues to the drug-sensitivity of Eg5. (B) Steady-state enzymatic parameters for engineered human Eg5 motor domain mutants. Steady-state ATP hydrolysis by Eg5 was measured using an NADH-enzyme coupled assay varying the concentration of ATP, microtubules, or monastrol to obtain the microtubule concentration for half-maximal stimulation (K_0.5MT), the maximal rate of microtubule-stimulated ATP hydrolysis (k_cat), and the concentration of monastrol that inhibits basal ATP hydrolysis by 50 % (IC₅₀), as described [28].