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首页> 外文期刊>Biochemistry >Mutant Cholinesterases Possessing Enhanced Capacity for Reactivation of Their Phosphonylated Conjugates.
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Mutant Cholinesterases Possessing Enhanced Capacity for Reactivation of Their Phosphonylated Conjugates.

机译：突变型胆碱酯酶具有增强的磷酸化缀合物再活化的能力。

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Selective mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7) phosphonylated with chiral S(P)- and R(P)-cycloheptyl, -3,3-dimethylbutyl, and -isopropyl methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE and butyrylcholinesterase (EC 3.1.1.8). Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29000 times) as a reactivator of S(P)-phosphonates (k(r) ranged from 50 to 13000 min(-)(1) M(-)(1)), while R(P) conjugates were reactivated by both oximes at similar, but far slower, rates (k(r) < 10 min(-)(1) M(-)(1)). The Y337A substitution accelerated all reactivation rates over the wild-type AChE and enabled reactivation even of R(P)-cycloheptyl and R(P)-3,3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of reactivation rates of the S(P) conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, S(P)-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure.
机译：通过肟HI-重新激活被手性S（P）-和R（P）-环庚基，-3,3-二甲基丁基和-异丙基甲基膦酰基硫代胆碱磷酸化的小鼠乙酰胆碱酯酶（AChE; EC 3.1.1.7）的选择性突变体与野生型AChE和丁酰胆碱酯酶（EC 3.1.1.8）相比，图6和2-PAM及其再活化动力学。胆碱结合位点的突变（Y337A，Y337A / F338A）或与酰基口袋突变（F295L / Y337A，F297I / Y337A，F295L / F297I / Y337A）结合使用以扩大活性中心峡谷的尺寸。 HI-6作为S（P）-膦酸酯（k（r）的活化剂，范围从50到13000 min（-）（1）M（-）（1）比2-PAM更有效（高达29000次） ），虽然R（P）共轭物被两种肟以相似但又慢得多的速率重新激活（k（r）<10 min（-）（1）M（-）（1））。 Y337A取代加速了野生型AChE的所有再活化速率，甚至使R（P）-环庚基和R（P）-3,3-二甲基丁基共轭物（在野生型AChE中形成对再活化具有抗性）也能再活化。当与酰基囊中的F295L或F297I突变结合使用时，Y337A突变显示S（P）共轭物的再激活率显着提高。 HI-6使F295L / Y337A被最庞大的烷氧基部分S（P）-环庚基甲基膦酸酯化的F295L / Y337A进行了120倍的最大增强。该显着增强可能是同时增加胆碱结合位点和酰基袋的尺寸的直接结果。如分子模型所建议的那样，尺寸的增加允许优化在空间影响的峡谷中肟的进攻角度。重新活化的速率达到足以考虑将突变酶和肟的混合物作为保护和处理有机磷酸盐暴露的清除策略的值。

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