D'Ordine R L, Bahnson B J, Tonge P J, Anderson V E
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Biochemistry. 1994 Dec 13;33(49):14733-42. doi: 10.1021/bi00253a011.
3-Quinuclidinone catalyzes the exchange of the alpha-protons of butyryl-coenzyme A (CoA) with a second-order rate constant of 2.4 x 10(-6) M-1 s-1. In contrast, enoyl-CoA hydratase catalyzes the stereospecific exchange of the pro-2S proton of butyryl-CoA with a maximum second-order rate constant of ca. 8 x 10(2) M-1 s-1. This isotope exchange reaction is completely stereospecific within the limits of experimental detection (over 600-fold). The enzyme-catalyzed exchange is dependent on pD, decreasing above a pKa of 8.8 and below a pKa of 8.1, but independent of the buffer concentration. The stereospecificity of the exchange was unexpected because the pro-2R hydrogen is abstracted during the enzyme-catalyzed dehydration of 3(S)-hydroxybutyryl-CoA. In spite of the ability to exchange the pro-2S hydrogen, the stereospecificity of the dehydration reaction was determined to be better than 1 in 10(5) as no incorporation of 2H into the alpha-position of crotonyl-CoA or into the pro-2S position of 3(S)-hydroxybutyryl-CoA was detected during prolonged equilibrations with enoyl-CoA hydratase. Both the exchange of the alpha-proton and the dehydration activity of the enzyme are diminished by over 100-fold in a site-directed mutation of rat liver enoyl-CoA hydratase, where glutamate-164 is changed to glutamine, strongly suggesting that the same active site base is responsible for proton abstraction in both the dehydration and solvent exchange reactions. The enoyl-CoA hydratase-catalyzed exchange of the alpha-protons becomes nonstereospecific when the acidity of the alpha-protons is enhanced. While alpha-proton abstraction can be observed when no elimination reaction is possible, there is no evidence for proton abstraction without elimination in the crotonase equilibrations with 3(S)-hydroxybutyryl-CoA, 3-hydroxypropionyl-CoA, or 3-chloropropionyl-CoA. The differences in the isotope exchange and dehydration reactions emphasize the importance of the 3-hydroxyl group in promoting elimination and are consistent with a concerted elimination mechanism.
3-奎宁环酮催化丁酰辅酶A（CoA）的α-质子交换，二级速率常数为2.4×10⁻⁶ M⁻¹ s⁻¹。相比之下，烯酰辅酶A水合酶催化丁酰辅酶A的pro-2S质子的立体特异性交换，最大二级速率常数约为8×10² M⁻¹ s⁻¹。在实验检测范围内（超过600倍），这种同位素交换反应是完全立体特异性的。酶催化的交换依赖于pD，在pKa高于8.8和低于8.1时降低，但与缓冲液浓度无关。交换的立体特异性出乎意料，因为在酶催化的3(S)-羟基丁酰辅酶A脱水过程中，pro-2R氢被提取。尽管能够交换pro-2S氢，但脱水反应的立体特异性被确定优于10⁵分之一，因为在用烯酰辅酶A水合酶长时间平衡过程中，未检测到2H掺入巴豆酰辅酶A的α-位或3(S)-羟基丁酰辅酶A的pro-2S位。在大鼠肝脏烯酰辅酶A水合酶的定点突变中，谷氨酸-164变为谷氨酰胺，酶的α-质子交换和脱水活性均降低了100倍以上，这强烈表明相同的活性位点碱负责脱水和溶剂交换反应中的质子提取。当α-质子的酸度增强时，烯酰辅酶A水合酶催化的α-质子交换变得非立体特异性。虽然在不可能发生消除反应时可以观察到α-质子提取，但在与3(S)-羟基丁酰辅酶A、3-羟基丙酰辅酶A或3-氯丙酰辅酶A的巴豆酸酶平衡中，没有证据表明没有消除反应就有质子提取。同位素交换和脱水反应的差异强调了3-羟基在促进消除中的重要性，并与协同消除机制一致。
