Skip to main content
. 2022 Dec 31;299(2):102855. doi: 10.1016/j.jbc.2022.102855

Figure 2.

Figure 2

tMTHFR•CHO-H4folate complex formation and effects of pH and folate on tMTHFR Activity.A, UV-VIS spectra of the tMTHFR•CHO-H4folate complex. UV-VIS spectra of tMTHFR with (blue) or without (red) CHO-H4folate treatment. The solid black line in the inset corresponds the calculated spectrum resulting from subtracting the spectrum of folate-free tMTHFR (red line, as a reference) from that of the tMTHFR•CHO-H4folate complex (blue line). The dotted green line represents a free CHO-H4folate spectrum. B, effect of pH on NADH:menadione oxidoreductase activity. The tMTHFR activity was measured in varied pH units and at room temperature. Equation 1 was used to fit data (43). The vertical axis represents the percentage of the uninhibited initial velocity. Relative activities of tMTHFR at pH 6.0 (red circles), 7.0 (blue squares), and 8.0 (green diamonds) in the presence of varying concentrations of free CHO-H4folate are shown (43). The bars represent the SD of triplicate measurements. C, double-reciprocal plots of NADH-menadione oxidoreductase activities of tMTHFR in the presence of (6RS)-CHO-H4folate. Relative activities of tMTHFR in the presence of varying concentrations of free CHO-H4folate are shown: at 0 μM (closed circle), 1 μM (open square), 5 μM (open circle), and 20 μM (6S)-CHO-H4folate (closed square) respectively. D, effect of pH on the CH3-H4folate:menadione oxidoreductase assay. tMTHFR activity, measured by the formation of CH+ = H4folate as a function of increasing CH3-H4folate concentration, was performed in triplicate at 50 °C. The bars represent the SD of triplicate measurements. Equation 2 was used to fit data (34). Color scheme is the same as in Panel B. MTHFR, methylenetetrahydrofolate reductase; tMTHFR, Thermus thermophilus MTHFR.