Porter D J, Bright H J
J Biol Chem. 1980 May 25;255(10):4772-80.
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.
我们描述了3-硝基丙酸酯、3-硝基-2-羟基丙酸酯和3-硝基-2-氨基丙酸酯的3-碳负离子（I⁻）和3-碳共轭酸（I）在25℃下与延胡索酸酶和天冬氨酸酶的相互作用。（化学式：见正文）1. Ia⁻和Ib⁻竞争性抑制延胡索酸酶，与Ia和Ib相比，它们的结合紧密程度分别高出4000倍和18000倍以上，与琥珀酸相比，分别高出5000倍和11000倍以上。Ic竞争性抑制天冬氨酸酶，与Ic相比，其结合紧密程度（取决于2-氨基的电离状态）高出220至1630倍以上，与琥珀酸相比，高出290至2200倍以上。2. 延胡索酸酶和天冬氨酸酶与I⁻的结合比它们各自的底物紧密得多。因此，延胡索酸酶的Km/KIa⁻和Km/KIb⁻分别为375和900（在pH 7.0时，KIa⁻ = 64 nM，KIb⁻ = 27 nM）。对于天冬氨酸酶，Km/KIa⁻ = 519，而Km/KIc⁻ = 1630（2-氨基未质子化）或220（2-氨基质子化）。3. 在pH 7.0时，延胡索酸酶的kI和k-I（化学式：见正文）值，对于Ia⁻分别为0.55×10⁸ M⁻¹ s⁻¹和3.5 s⁻¹，对于Ib⁻分别为2.6×10⁸ M⁻¹ s⁻¹和6.9 s⁻¹。这些结果，连同苹果酸酶对照实验的结果表明，Ia⁻、Ib⁻和Ic⁻是过渡态或瞬态中间体类似物，并且延胡索酸酶和天冬氨酸酶反应的机制涉及酶结合的3-碳负离子。
