Aspartate 120 of Escherichia coli Methylenetetrahydrofolate Reductase: Evidence for Mojor Roles in Folate Binding and Catalysis and a Minor Role in Flavin Reactivity.

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 NI -C20 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 (kcat/Km) in the CH2-H4- folate 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 Kd of up to 15-fold were obtained for the mutants. Examining the Ered + 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 Asp l2OAla mutant, but not the severely impaired Asp l2OLys mutant, demonstrated the species, suggesting a connection between the extent of complex formation and catalytic efficiency. [ABSTRACT FROM AUTHOR]