Millard C B, Lockridge O, Broomfield C A
United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5425, USA.
Biochemistry. 1995 Dec 12;34(49):15925-33. doi: 10.1021/bi00049a007.
Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) nerve agents. To overcome this limitation, we selected several residues that were predicted to be within 3-10 A of both the active site Ser O gamma and the oxyanion hole of human butyrylcholinesterase for mutation to His (G115H, G117H, Q119H, and G121H). In remarkable contrast with wild-type (WT) and all other His mutants tested, G117H underwent spontaneous reactivation following OP inhibition to regain 100% of original esterase activity with maximum k3 values of approximately 6.8 x 10(-5) and 16 x 10(-5) s-1 for GB (sarin) and VX, respectively, in 0.1 M Bis-Tris, 25 degrees C. The free energy of activation for k3 was 19 kcal mol-1, and measurement of pH dependence suggested that reactivation resulted from an acidic group with pKa 6.2. To evaluate further the importance of His in achieving this result, we changed the same Gly to Lys (G117K) and compared its substrate and inhibitor kinetics with those of G117H. Both mutants retained esterase activity with Km values similar to those of WT for neutral ester hydrolysis, but G117K did not reactivate. Complete reactivation proves that G117H is not irreversibly inhibited but instead functions as a catalyst for OP hydrolysis. Dephosphonylation is the rate-limiting step, and G117H effects overall rate constant enhancements of approximately 100- and 2000-fold above the uncatalyzed hydrolysis of GB and VX, respectively, at pH 6.0, 25.0 degrees C. We conclude that an appropriately positioned imidazolium ion in the oxyanion hole catalyzes dephosphonylation and, thereby, confers a novel organophosphorus acid anhydride hydrolase activity upon butyrylcholinesterase.
丝氨酸酯酶和蛋白酶会被有机磷（OP）神经毒剂迅速且不可逆地抑制。为克服这一局限性，我们选择了几个预计位于人丁酰胆碱酯酶活性位点丝氨酸Oγ和氧阴离子洞3 - 10埃范围内的残基，将其突变为组氨酸（G115H、G117H、Q119H和G121H）。与野生型（WT）以及测试的所有其他组氨酸突变体形成显著对比的是，G117H在被OP抑制后会自发重新激活，在0.1 M Bis - Tris、25℃条件下，对于GB（沙林）和VX，分别以约6.8×10⁻⁵和16×10⁻⁵ s⁻¹的最大k3值恢复100%的原始酯酶活性。k3的活化自由能为19 kcal mol⁻¹，对pH依赖性的测量表明重新激活是由pKa为6.2的酸性基团导致的。为进一步评估组氨酸在实现这一结果中的重要性，我们将相同的甘氨酸替换为赖氨酸（G117K），并将其底物和抑制剂动力学与G117H进行比较。两个突变体都保留了酯酶活性，对于中性酯水解，其Km值与WT相似，但G117K没有重新激活。完全重新激活证明G117H不是被不可逆地抑制，而是作为OP水解的催化剂起作用。去磷酸化是限速步骤，在pH 6.0、25.0℃时，G117H使GB和VX的总体速率常数分别比未催化水解提高约100倍和2000倍。我们得出结论，氧阴离子洞中位置合适的咪唑鎓离子催化去磷酸化，从而赋予丁酰胆碱酯酶一种新的有机磷酸酸酐水解酶活性。
