title: 3-Carbanionic substrate analogues bind very tightly to fumarase and aspartase.
abstract: 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.
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000642:Ammonia-Lyases / Q000378:metabolism*; D000818:Animals; D001220:Aspartate Ammonia-Lyase / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology; D002645:Chickens; D005649:Fumarate Hydratase / Q000378:metabolism*; D007477:Ions; D007700:Kinetics; D007773:Lactates / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000378:metabolism; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology; D009574:Nitro Compounds; D011422:Propionates / Q000494:pharmacology*; D011485:Protein Binding; D013552:Swine
chemical_list: C024847:3-nitro-2-aminopropionic acid; C024846:3-nitro-2-hydroxypropionate; D007477:Ions; D007773:Lactates; D009574:Nitro Compounds; D011422:Propionates; D008291:Malate Dehydrogenase; D005649:Fumarate Hydratase; D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase; D000409:Alanine; C015392:3-nitropropionic acid