3-Carbanionic substrate analogues bind very tightly to fumarase and aspartase.

We describe the interactions at 25 degrees C of the 3-carbanions (I-) and 3-carbon conjugate acids (I) of 3-nitropropionate, 3-nitro-2-hydroxypropionate, and 3-nitro-2-aminopropionate with fumarase and aspartase. (Formula: see text) 1. Ia- and Ib- inhibit fumarase competitively and are bound more than 4000- and 18,000-fold more tightly, respectively, than Ia and Ib, and 5000- and 11,000-fold more tightly, respectively, than succinate. Ic inhibits aspartase competitively and is bound (depending on the ionization state of the 2-amino group) more than 220- to 1630-fold more tightly than Ic and 290- to 2200-fold more tightly than succinate. 2. Fumarase and asparatse bind I- much more tightly than their respective substrates. Thus, Km/KIa- and Km/KIb- for fumarase are 375 and 900, respectively (with KIa- = 64 nm and KIb- = 27 nM at pH 7.0). For asparatse, Km/KIa- = 519, while Km/KIc- = 1630 (2-amino group unprotonated) or 220 (2-amino group protonated). 3. The values of kI and k-I (FORMULA: SEE TEXT) for fumarase are respectively, 0.55 X 10(8) M-1 s-1 and 3.5 s-1 for Ia- and 2.6 X 10(8) M-1 s-1 and 6.9 s-1 for Ib- at pH 7.0. These results, together with those of control experiments with Malic Enzyme, suggest that Ia-, Ib-, and Ic- are transition state, or transient intermediate, analogues and that the mechanisms of the fumarase and aspartase reactions involve enzyme-bound 3-carbanions.