PH‐dependent studies on the reductive half‐reaction of E. coli methylenetetrahydrofolate reductase (769.13)

Abstract

Methylenetetrahydrofolate reductase (MTHFR) is a flavoprotein that utilizes two substrates, NADH and 5,10‐methylenetetrahydrofolate (CH2‐H4folate), in a ping pong bi‐bi mechanism. In the reaction, bound FAD is reduced by NADH and then oxidized by CH2‐H4folate. The pH dependence of the reductive half‐reaction was studied with steady‐state kinetics using the NADH‐menadione oxidoreductase assay. In this assay the reductive half‐reaction is rate‐limiting, and menadione is used to reoxidize the flavin. The wild‐type MTHFR reaction was found to be pH‐dependent with an inflection point (pKa) at 4.9. We have hypothesized that this pKa belongs to the active‐site amino acid glutamate 28 (Glu28). When Glu28 is deprotonated, the negatively‐charged side chain can stabilize the positively‐charged nicotinamide ring of NADH during the reductive half‐reaction. To determine whether Glu28 is responsible for the pKa of 4.9, we have examined a mutant enzyme with Glu28 replaced by a glutamine. Kinetic parameters were obtained for the NADH‐menadione reaction catalyzed by the Glu28Gln mutant at pH values 4.5 to 8. Our results suggest that a pKa ~5 still exists in the pH rate profile of the Glu28Gln mutant. However, instability and denaturation have plagued our studies. In current work, we are testing alternate buffers conditions to stabilize the mutant enzyme.