Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli.

The kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been investigated by using progress curve, initial velocity, product inhibition, and dead-end inhibition studies as well as isotope effects. The results indicate that the reaction conforms to a random mechanism involving two dead-end complexes, viz., enzyme-DHF-THF and enzyme-NADP-DHF. At higher concentrations, DHF causes substrate inhibition by combining at the NADPH binding site on the enzyme. The steady-state velocity data can be analyzed adequately on the basis that rapid-equilibrium conditions apply. However, this can be only an approximate description of the reaction since the isotope effects observed with NADPD demonstrate clearly that catalysis cannot be rate limiting at pH 7.4. The choice of conditions for analysis of progress-curve data is discussed in the Appendix.