Kinetics of substrate, coenzyme, and inhibitor binding to Escherichia coli dihydrofolate reductase

Abstract

NADPH, folate, dihydrofolate and the inhibitors trimethoprim and methotrexate bind rapidly and reversibly to both dihydrofolate reductase isoenzymes, which are isolated from E. coli RT500. The coenzyme and substrates appear to bind to only 1 of the mixture of 2 forms of the isoenzyme present at equilibrium, while the inhibitors bind to both forms. The proportions of the 2 forms are different for the 2 isoenzymes and are pH dependent in each case. The measured association rate constants for substrates and inhibitors lie in the range (1-2) .times. 107 M-1 s-1 at 25.degree. C, but are unlikely to be diffusion controlled. The rate constant for NADPH binding is 2 .times. 106 M-1 s-1. The formation of binary complexes takes place through a multistep mechanism. A minimum of 3 steps is required to explain the kinetic results. An equilibrium between 2 or more forms of the enzyme-ligand complex governs the overall dissociation. The stability of this equilibrium is largely responsible for the tighter binding of inhibitors relative to substrate or coenzyme, and also for the different binding strengths of inhibitors to the isoenzymes.