Aspartate 120 of Escherichia coli Methylenetetrahydrofolate Reductase: Evidence for Major Roles in Folate Binding and Catalysis and a Minor Role in Flavin Reactivity
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- 作者：Elizabeth E. Trimmer ; David P. Ballou ; Lara J. Galloway ; Sara A. Scannell ; Danielle R. Brinker ; and Katie R. Casas
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：May 10, 2005
- 年：2005
- 卷：44
- 期：18
- 页码：6809 - 6822
- 全文大小：218K
- 年卷期：v.44,no.18(May 10, 2005)
- ISSN：1520-4995
文摘
Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADH-linkedreduction of 5,10-methylenetetrahydrofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate)using flavin adenine dinucleotide (FAD) as cofactor. MTHFR is unusual among flavin oxidoreductasesbecause it contains a conserved, negatively rather than positively charged amino acid (aspartate 120) nearthe N1-C2=O position of the flavin. At this location, Asp 120 is expected to influence the redox propertiesof the enzyme-bound FAD. Modeling of the CH3-H4folate product into the enzyme active site suggeststhat Asp 120 may also play crucial roles in folate binding and catalysis. We have replaced Asp 120 withAsn, Ser, Ala, Val, and Lys and have characterized the mutant enzymes. Consistent with a loss of negativecharge near the flavin, the midpoint potentials of the mutants increased from 17 to 30 mV. A smallkinetic 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 (kcat/Km) in the CH2-H4folate oxidative half-reaction was decreased significantly (up to 70000-fold) and in a manner generallyconsistent with the negative charge density of position 120, supporting a major role for Asp 120 inelectrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. Asp 120 is alsointimately involved in folate binding as increases in the apparent Kd of up to 15-fold were obtained forthe mutants. Examining the Ered + CH2-H4folate reaction at 4 
C, we obtained, for the first time, evidencefor the rapid formation of a reduced enzyme-folate complex with wild-type MTHFR. The more activeAsp120Ala mutant, but not the severely impaired Asp120Lys mutant, demonstrated the species, suggestinga connection between the extent of complex formation and catalytic efficiency.
C, we obtained, for the first time, evidencefor the rapid formation of a reduced enzyme-folate complex with wild-type MTHFR. The more activeAsp120Ala mutant, but not the severely impaired Asp120Lys mutant, demonstrated the species, suggestinga connection between the extent of complex formation and catalytic efficiency.