The origin of the electrostatic perturbation in acetoacetate decarboxylase
The origin of the electrostatic perturbation in acetoacetate decarboxylase
复制标题
DOI：
10.1038/nature07938
复制
发表时间：
2009-05-21
期刊：
影响因子：
64.8
通讯作者：
Allen, Karen N.
中科院分区：
文献类型：
--
作者：
Ho, Meng-Chiao;Menetret, Jean-Francois;Allen, Karen N.
乙酰乙酸脱羧酶（AAD酶）长期以来一直被认为是酶活性位点中可电离基团的pK（a）值发生显著变化的典型例子。在1966年，有人假设在AAD酶中，在亲核性Lys 115中观察到的大pK（a）扰动（-4.5 log单位）的起源是由于Lys 116的接近，这标志着酶学中微环境效应的第一个提议。静电扰动假说已经在许多酶中得到了证实，但由于缺乏三维结构，从未在激发其概念的酶中得到证实。在这里，我们提出的X-射线晶体结构的AAD酶和烯胺加合物与底物类似物2，4-戊二酮。令人惊讶的是，Lys 115的pK（a）的偏移不是由于Lys 116的接近，其侧链远离活性位点取向。相反，赖氨酸116参与赖氨酸115在由酶的新折叠提供的长的疏水漏斗中的结构锚定。因此，AAD酶通过将侧链置于蛋白质核心中同时加强极性残基的邻近性的去溶剂化效应来扰乱亲核试剂的pK（a），这通过静电和空间效应促进脱羧。
Acetoacetate decarboxylase (AADase) has long been cited as the prototypical example of the marked shifts in the pK(a) values of ionizable groups that can occur in an enzyme active site. In 1966, it was hypothesized that in AADase the origin of the large pK(a) perturbation (-4.5 log units) observed in the nucleophilic Lys 115 results from the proximity of Lys 116, marking the first proposal of microenvironment effects in enzymology. The electrostatic perturbation hypothesis has been demonstrated in a number of enzymes, but never for the enzyme that inspired its conception, owing to the lack of a three-dimensional structure. Here we present the X-ray crystal structures of AADase and of the enamine adduct with the substrate analogue 2,4-pentanedione. Surprisingly, the shift of the pK(a) of Lys 115 is not due to the proximity of Lys 116, the side chain of which is oriented away from the active site. Instead, Lys 116 participates in the structural anchoring of Lys 115 in a long, hydrophobic funnel provided by the novel fold of the enzyme. Thus, AADase perturbs the pK(a) of the nucleophile by means of a desolvation effect by placement of the side chain into the protein core while enforcing the proximity of polar residues, which facilitate decarboxylation through electrostatic and steric effects.