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A Proposed Proton Relay Mechanism in Methylenetetrahydrofolate Reductase

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10‐methylenetetrahydrofolate (CH2‐H4folate) to 5‐methyltetrahydrofolate (CH3‐H4folate) by NAD(P)H. This reaction can be divided into two half‐reactions: the NAD(P)H reductive halfreaction and the folate oxidative half‐reaction. The X‐ray crystal structure of the E. coli enzyme has revealed a hydrogen‐bonded network of three active site amino acids, Glu28, Ser26 and His273. Previous studies with a Glu28Gln mutant indicated that Glu28, located near N10 of CH3‐H4folate, is an essential amino acid. Since Ser26 and His273 form hydrogen bonds to Glu28, we propose that Ser26, His273 and Glu28 are involved in a proton relay mechanism which donates a proton to CH2‐H4folate in the folate oxidative half‐reaction. In order to elucidate the role of Ser26 in this mechanism, we have characterized Ser26Thr and Ser26Ala mutants using the steady‐state CH3‐H4folate‐menadione oxidoreductase assay, in which the folate oxidative half‐reaction is rate limiting. We have hypothesized that the Ser26Thr mutant will have activity comparable to the wild‐type enzyme and the Ser26Ala mutant will have reduced activity due to its inability to hydrogen bond. Contrary to our hypothesis, we show that both mutants catalyze the folate reaction similar to the wild‐type enzyme. We also demonstrate that the Ser26Ala mutant shows competitive substrate inhibition in the CH3‐H4folate‐menadione assay, which is not observed for the wild‐type enzyme. Examination of the mutants in the NADH‐menadione assay shows kinetic parameters similar to those of the wild‐type enzyme. Taken together, our results suggest that Ser26 may not play an important role in either folate or NADH catalysis of MTHFR. However, we propose that in the case of the folate reaction, a water molecule inserted between the alanine and His273 might function like a serine in the proton relay mechanism, explaining the lack of a defect in the Ser26Ala mutant. To investigate the proton relay mechanism and the role of Ser26 further, we are studying the pH‐dependency of MTHFR in the CH3‐H4folate‐menadione assay.