Catalytic mechanism of the dihydrofolate reductase reaction as determined by pH studies.

The variation with pH of the kinetic parameters of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been determined with the aim of elucidating the chemical mechanism of the reaction. The (V/K)DHF and V profiles indicated that protonation enhances the observed rate of interaction of dihydrofolate (DHF) with the enzyme-NADPH complex as well as the maximum velocity of the reaction. The pKa value of 8.09 observed in the (V/K)DHF profile is similar to that of 7.9 observed in the Ki profile for 2,4-diamino-6,7-dimethylpteridine while the pKa value of the V profile is displaced to 8.4. From the magnitude of the pH-independent value for (V/K)DHF, it is concluded that unprotonated dihydrofolate must react, at neutral pH, with the protonated form of the enzyme. The D(V/K)DHF value is independent of pH and equal to unity whereas the DV value varies as a wave function of pH with limiting values of 1.5 and 1.0 at low and high pH, respectively. It is proposed that dihydrofolate reacts with the unprotonated enzyme-NADPH complex to form a dead-end complex and with the protonated form of the same complex to form a productive complex. Further, it is considered that the protonated carboxyl of Asp-27 at the active site of the enzyme is responsible for the protonation of the N-5 nitrogen of dihydrofolate and that this protonation precedes and facilitates hydride transfer.