Kinetics of methotrexate binding to dihydrofolate reductase from Neisseria gonorrhoeae

The kinetics of methotrexate inhibition of dihydrofolate reductase from Neisseria gonorrhoeae have been investigated. Methotrexate was shown to be a tight-binding inhibitor ( K i = 13 pM) competitive with dihydrofolate. However, “stoichiometric” or “pseudoirreversible“ inhibition could not be demonstrated. Progress curves of inhibited assays quickly attained steady state regardless of the order of substrate addition, indicating that methotrexate association and dissociation processes were rapid. Kinetic techniques were used to measure the rate of methotrexate dissociation from the enzyme-NADPH-methotrexate ternary complex. At 30°, the first-order off-rate constant ( k off ) was calculated to be 0.56 min −1 . This value is ∼40-fold greater than the dissociation rate constant of methotrexate for Escherichia coli dihydrofolate reductase. At lower temperatures, progress curves of methotrexate-inhibited gonococcal enzyme assays displayed marked increases in both curvature and the time to reach steady state. At 9°, the methotrexate dissociation rate was slow enough ( k off = 0.04 min −1 ) so that initial velocities of the reaction could be measured, and under these conditions methotrexate inhibition was shown to be “stoichiometric”.