Chemical mechanism of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium: pH studies and chemical modification.

Abstract

The variation with pH of the kinetic parameters associated with dihydrofolate reductase from Streptococcus faecium has been used to gain information about the chemical mechanism of the reaction catalyzed by the enzyme. The pH dependence of log V/K for dihydrofolate showed that a group with a pK value of 4.7 must be ionized and that a group with a pK value of 6.6 must be protonated for activity. Temperature and solvent perturbation studies indicate that these groups are probably the carboxyls of the glutamate moiety of dihydrofolate and of an aspartate residue on the enzyme, respectively. The similarity of the pH profile and the magnitude of the pK value for the linear competitive inhibitor 2,4-diaminopteridine suggest that the carboxyl group is concerned with the binding of dihydrofolate and its analogues to the enzyme. This conclusion is confirmed by the result that a group with a pK value of 6.7 must be protonated for the binding of methotrexate. It is proposed that the binding involves the formation with N-5 of dihydrofolate or N-1 of methotrexate of a hydrogen bond which has considerable ionic character and which lies within a hydrophobic environment. Further, it is suggested that the same hydrogen acts as an auxiliary catalyst which facilitates hydride transfer from NADPH to dihydrofolate for its conversion to tetrahydrofolate. Evidence to support this suggestion comes from the finding that the V profile is similar to the V/K profile except that the pK of the group which must be protonated for maximum enzyme activity is shifted upward by about 2 pH units. Such an increase in a pK value is consistent with the formation of a hydrogen ionic bond in the ternary enzyme-NADPH-dihydrofolate complex. The results of inactivation experiments with trinitrobenzenesulfonate appear to indicate that a lysine residue is necessary to maintain the enzyme in its active conformation.