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pH‐dependent Studies of E. coli Methylenetetrahydrofolate Reductase
Author(s) -
Wang Jingjing,
Trimmer Elizabeth E.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.756.13
Subject(s) - menadione , chemistry , deprotonation , methylenetetrahydrofolate reductase , reductase , flavoprotein , biochemistry , stereochemistry , enzyme , steady state (chemistry) , cofactor , organic chemistry , ion , genotype , gene
E. coli methylentetrahydrofolate reductase (MTHFR) is a flavoprotein that catalyzes the NADH‐ dependent reduction of 5,10‐methylenetetrahydrofolate (CH 2 ‐H 4 folate) to 5‐ methyltetrahydrofolate (CH 3 ‐H 4 folate). The pH‐dependency of MTHFR was studied in order to understand the roles of the ionizable active site amino acids in catalyzing the reaction. The reverse of the folate oxidative half‐reaction was studied at pH values 5–10 with the CH 3 ‐H 4 folate‐menadione steady‐state kinetics assay. In this assay, menadione acts as an alternate electron accepter to oxidize the reduced enzyme. Our results show that a basic condition (pH 7–10) is helpful for the reverse folate reaction. An inflection point around pH 6 may reflect the pK a of His273, an amino acid implicated in a proposed proton transfer cascade necessary for folate deprotonation. Currently, we are characterizing the pH dependence of the NADH half‐reaction using the NADH‐menadione assay and the physiological reaction using the NADH‐CH 2 ‐H 4 folate steady‐state assay. Research was supported by HHMI grant #52006298 to Grinnell College.