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The minor role of Gln 183 in the NADH reductive half‐reaction of E. coli methylenetetrahydrofolate reductase (769.10)
Author(s) -
Zuo Chong,
Jolly Amber,
Nikolova Andriana,
Cao Sirui,
Sanchez Jeremy,
Trimmer Elizabeth
Publication year - 2014
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.28.1_supplement.769.10
Subject(s) - chemistry , flavoprotein , cofactor , reductase , oxidoreductase , enzyme , menadione , active site , stereochemistry , enzyme kinetics , flavin group , redox , methylenetetrahydrofolate reductase , biochemistry , organic chemistry , genotype , gene
The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10‐methylenetetrahydrofolate (CH 2 ‐H 4 folate) to 5‐methyltetrahydrofolate (CH 3 ‐H 4 folate) using FAD as a redox coenzyme. The reaction contains two different half‐reactions, the NADH reductive half‐reaction and the CH 2 ‐H 4 folate oxidative half‐reaction. The focus of our work is the active‐site amino acid Glutamine 183 (Gln183). Structural studies have implicated the importance of Gln183 in both substrate binding and catalysis of the two half‐reactions, based on direct hydrogen bonds between Gln183 and the substrates NADH and folate. In order to investigate the function of Gln183 in MTHFR catalysis, two mutants with single amino acid changes, Gln183Ala (Q183A) and Gln183Glu (Q183E) have been constructed. Using the steady‐state NADH‐menadione oxidoreductase assay, we have examined the reductive half‐reactions of each mutant enzyme. For reasons of mutant stability, the temperature of the assay was optimized to 4°C. We expected that the change in hydrogen bonds and polarity of the Q183 mutant enzymes would affect their kinetic parameters k cat and K m . However, the mutant enzymes have similar kinetic parameters to the wild‐type enzyme at 4°C, consistent with our previous results at 25°C. Therefore, we conclude that glutamine 183 plays only a minor role in the reductive half‐reaction of MTHFR. Grant Funding Source : Grinnell College