Figure 1.
Chemical structures of fumarate and its analogs.
These structures were generated using Accelrys Draw (Accelrys, USA).
Figure 2.
Allosteric activation of human m-NAD(P)-ME by fumarate and its analogs.
(A) Fumarate; (B) Mesaconate; (C) Trans-aconitate; (D) Monomethyl fumarate; (E) Monoethyl fumarate. Closed circles, m-NAD(P)-ME; open circles, m-NAD(P)-ME_R67A/R91A; closed triangles, m-NAD(P)-ME_K57S/E59N/K73E/D102S; open triangles, c -NADP-ME. The specific activities of the m-NADP-(P)-ME WT, R67A/R91A, K57S/E59N/K73E/D102S and c-NADP-ME WT were approximately 0.1, 0.005, 0.02 and 0.08 µmol/min, respectively, and the final enzyme concentration in an individual assay was 20, 400, 100, and 25 ng/µl, respectively. The v and v0 represented the enzyme activity in the presence and absence of fumarate analogs, respectively.
Table 1.
Effect of fumarate analogs on human m-NAD(P)-ME and c-NADP-ME1,2.
Figure 3.
Allosteric inhibition of human m-NAD(P)-ME by fumarate and its analogs.
(A) Dimethyl fumarate; (B) Diethyl fumarate; (C) Oxaloacetate; (D) Diethyl oxalacetate; (E) Dimethyl maleate; (F) Diethyl maleate. Closed circles, m-NAD(P)-ME; open circles, m-NAD(P)-ME_R67A/R91A; closed triangles, m-NAD(P)-ME_K57S/E59N/K73E/D102S; open triangles, c-NADP-ME. The specific activities of the m-NADP-(P)-ME WT, R67A/R91A, K57S/E59N/K73E/D102S and c-NADP-ME WT were approximately 0.1, 0.005, 0.02 and 0.08 µmol/min, respectively, and the final enzyme concentration in an individual assay was 20, 400, 100, and 25 ng/µl, respectively. The v and v0 represented the enzyme activity in the presence and absence of fumarate analogs, respectively.
Figure 4.
Effect of fumarate on diethyl oxalacetate-inhibited m-NAD(P)-ME activity.
(A) The diethyl oxalacetate inhibition experiment of WT m-NAD(P)-ME without (closed circles) or with (open circles) 5 mM fumarate. (B) The fumarate rescue experiment of WT m-NAD(P)-ME. The enzyme was preincubated with 3 mM diethyl oxalacetate, and then the activity was restored with increasing concentrations of fumarate. The specific activities of the m-NADP-(P)-ME WT was approximately 0.1 µmol/min and the final enzyme concentration in an individual assay was 20 ng/µl. The v and v0 represented the enzyme activity in the presence and absence of fumarate analogs, respectively.