Miller Stephen P, Gonçalves Susana, Matias Pedro M, Dean Antony M
Biotechnology Institute, The University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108 (USA).
Chembiochem. 2014 May 26;15(8):1145-53. doi: 10.1002/cbic.201400040. Epub 2014 May 2.
An active site lysine essential to catalysis in isocitrate dehydrogenase (IDH) is absent from related enzymes. As all family members catalyze the same oxidative β-decarboxylation at the (2R)-malate core common to their substrates, it seems odd that an amino acid essential to one is not found in all. Ordinarily, hydride transfer to a nicotinamide C4 neutralizes the positive charge at N1 directly. In IDH, the negatively charged C4-carboxylate of isocitrate stabilizes the ground state positive charge on the adjacent nicotinamide N1, opposing hydride transfer. The critical lysine is poised to stabilize-and perhaps even protonate-an oxyanion formed on the nicotinamide 3-carboxamide, thereby enabling the hydride to be transferred while the positive charge at N1 is maintained. IDH might catalyze the same overall reaction as other family members, but dehydrogenation proceeds through a distinct, though related, transition state. Partial activation of lysine mutants by K(+) and NH4 (+) represents a throwback to the primordial state of the first promiscuous substrate family member.
异柠檬酸脱氢酶（IDH）催化作用所必需的活性位点赖氨酸在相关酶中并不存在。由于所有家族成员都在其底物共有的(2R)-苹果酸核心处催化相同的氧化β-脱羧反应，那么一种对其中一种酶至关重要的氨基酸并非在所有酶中都存在，这似乎很奇怪。通常情况下，氢化物转移至烟酰胺C4会直接中和N1处的正电荷。在IDH中，异柠檬酸带负电荷的C4-羧酸盐会稳定相邻烟酰胺N1上的基态正电荷，从而阻碍氢化物转移。关键的赖氨酸准备好去稳定——甚至可能使——在烟酰胺3-羧酰胺上形成的氧阴离子质子化，从而在维持N1处正电荷的同时使氢化物能够转移。IDH可能催化与其他家族成员相同的整体反应，但脱氢反应是通过一个独特但相关的过渡态进行的。赖氨酸突变体被K(+)和NH4(+)部分激活代表着向第一个混杂底物家族成员的原始状态的回归。
