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Article

Aspartate 120 of Escherichia coli Methylenetetrahydrofolate Reductase: Evidence for Major Roles in Folate Binding and Catalysis and a Minor Role in Flavin Reactivity^†^,^‡

Supporting Info

Elizabeth E. Trimmer,*^§ David P. Ballou, Lara J. Galloway,^§ Sara A. Scannell,^§ Danielle R. Brinker,^§ and Katie R. Casas^§

Department of Chemistry, Grinnell College, Grinnell, Iowa 50112-2036, and Department of Biological Chemistry and Biophysics Research Division, The University of Michigan, Ann Arbor, Michigan 48109-1055

Biochemistry, 2005, 44 (18), pp 6809–6822

DOI: 10.1021/bi0477236

Publication Date (Web): April 14, 2005

†

This work was supported in part by American Chemical Society Petroleum Research Fund Grant 39599-GB4 (E.E.T.), by Howard Hughes Medical Institute Undergraduate Biological Sciences Education Grant 71100-503702 (Grinnell College), and by National Institutes of Health Grants GM11106 and GM64711 (D.P.B.).

‡

This paper is dedicated to Vincent Massey (1926−2002).

To whom correspondence should be addressed. E-mail: trimmere@grinnell.edu. Phone: (641) 269-4398. Fax: (641) 269-4285.

Grinnell College.

The University of Michigan.

Abstract

Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADH-linked reduction of 5,10-methylenetetrahydrofolate (CH₂-H₄folate) to 5-methyltetrahydrofolate (CH₃-H₄folate) using flavin adenine dinucleotide (FAD) as cofactor. MTHFR is unusual among flavin oxidoreductases because it contains a conserved, negatively rather than positively charged amino acid (aspartate 120) near the N1−C2O position of the flavin. At this location, Asp 120 is expected to influence the redox properties of the enzyme-bound FAD. Modeling of the CH₃-H₄folate product into the enzyme active site suggests that Asp 120 may also play crucial roles in folate binding and catalysis. We have replaced Asp 120 with Asn, Ser, Ala, Val, and Lys and have characterized the mutant enzymes. Consistent with a loss of negative charge near the flavin, the midpoint potentials of the mutants increased from 17 to 30 mV. A small kinetic effect on the NADH reductive half-reaction was also observed as the mutants exhibited a 1.2−1.5-fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (k_cat/K_m) in the CH₂-H₄folate oxidative half-reaction was decreased significantly (up to 70000-fold) and in a manner generally consistent with the negative charge density of position 120, supporting a major role for Asp 120 in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. Asp 120 is also intimately involved in folate binding as increases in the apparent K_d of up to 15-fold were obtained for the mutants. Examining the E_red + CH₂-H₄folate reaction at 4 °C, we obtained, for the first time, evidence for the rapid formation of a reduced enzyme−folate complex with wild-type MTHFR. The more active Asp120Ala mutant, but not the severely impaired Asp120Lys mutant, demonstrated the species, suggesting a connection between the extent of complex formation and catalytic efficiency.