Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.
Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.
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DOI：
10.1021/ja3037367
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发表时间：
2012-10-03
影响因子：
15
通讯作者：
Baker, David
中科院分区：
文献类型：
--
作者：
Richter, Florian;Blomberg, Rebecca;Khare, Sagar D.;Kiss, Gert;Kuzin, Alexandre P.;Smith, Adam J. T.;Gallaher, Jasmine;Pianowski, Zbigniew;Helgeson, Roger C.;Grjasnow, Alexej;Xiao, Rong;Seetharaman, Jayaraman;Su, Min;Vorobiev, Sergey;Lew, Scott;Forouhar, Farhad;Kornhaber, Gregory J.;Hunt, John F.;Montelione, Gaetano T.;Tong, Liang;Houk, K. N.;Hilvert, Donald;Baker, David
亲核催化是加速酯和酰胺水解的一般策略。在天然活性位点中，催化二元组和三元组等亲核元素通常与氧阴离子空穴配对进行底物活化，但很难解析出这些元素的独立贡献或理解它们在进化过程中是如何出现的。在这里，我们通过使用催化二元体和氧阴离子孔计算设计人工催化剂来探索酯酶活性的最低要求。我们发现使用由主链 NH 基团而不是由侧链或桥接水分子形成的设计氧阴离子孔的成功率要高得多，并且通过将该基序与 Cys-His 二元体相结合，在不同的支架中获得了四种活性设计。经过活性位点优化后，最活跃的变体对对硝基苯酯的裂解表现出 400 M−1s−1 的催化效率 (kcat/KM)。动力学实验表明，活性位点半胱氨酸按照设计程序快速酰化，但随后酰基酶中间体的缓慢水解限制了总体催化效率。此外，所设计的酶的晶体结构中未正确形成半胱氨酸-组氨酸二联体。这些结果凸显了计算设计必须克服的挑战才能实现高水平的活动。
Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion-holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by sidechains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (kcat/KM) of 400 M−1s−1 for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.
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DOI：
10.1073/pnas.93.10.5043
发表时间：
1996-05-14
影响因子：
11.1
作者：
Kast, P;AsifUllah, M;Hilvert, D
通讯作者：
Hilvert, D
影响因子：
3.6
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CHU, SH;MAUTNER, HG
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MAUTNER, HG
DOI：
10.1073/pnas.83.11.3743
发表时间：
1986-06-01
影响因子：
11.1
作者：
BRYAN, P;PANTOLIANO, MW;POULOS, T
通讯作者：
POULOS, T
DOI：
10.1073/pnas.251555398
发表时间：
2001-12-04
影响因子：
11.1
作者：
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通讯作者：
Mayo, SL
影响因子：
3.6
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Kamerlin, Shina C. L.;Chu, Zhen T.;Warshel, A.
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Warshel, A.