The course of phosphorus in the reaction of N-acetyl-L-glutamate kinase, determined from the structures of crystalline complexes, including a complex with an AlF4- transition state mimic
The course of phosphorus in the reaction of N-acetyl-L-glutamate kinase, determined from the structures of crystalline complexes, including a complex with an AlF4- transition state mimic
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DOI：
10.1016/s0022-2836(03)00716-2
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发表时间：
2003-08-01
影响因子：
5.6
通讯作者：
Rubio, V
中科院分区：
文献类型：
--
作者：
Gil-Ortiz, F;Ramón-Maiques, S;Rubio, V
N-乙酰-L-谷氨酸激酶（NAGK）是氨基酸激酶家族中的一个结构范例，它催化ATP对N-乙酰-L-谷氨酸（NAG）的γ-COO-基团的磷酸化。我们测定了NAGK与MgADP、NAG和过渡态类似物AlF_4 ~-，与MgADP和NAG，以及与ADP和SO_4 ~（2-）形成的复合物的晶体结构。通过与MgAMPPNP-NAG复合物的结构比较，可以描述磷酰基转移过程中的三个连续步骤：开始时，进攻和离开的O原子和P原子不完全对齐，进攻O原子和P原子之间的距离为2.8埃，中间时，双锥中间体，几乎完全对齐，距离为2.3埃;并且，当转移完成时。转移发生在线路上，是强关联的，与Lys 8和Lys 217稳定的过渡态和离去基团，分别与Lys 61，与早期的建议，不参与。在所有复合物中发现的三个水分子与Asp 162和Mg一起发挥着至关重要的结构作用。两个富含甘氨酸的环（beta1-alphaA和beta2-alphaB）也非常重要，它们与配体一起在不同的复合物中移动，它们与配体形成氢键，将它们锁定在反应位置或稳定过渡态。活性位点太窄，不能容纳底物而不压缩反应基团，并且这种压缩应变似乎是NAGK催化机制的关键组成部分，并且可能是氨基酸激酶家族的其他酶如氨基甲酸酯激酶的催化机制的关键组成部分。两种底物的初始结合将需要具有更宽活性位点的不同酶构象，并且底物结合的能量将用于改变活性中心的构象，导致底物应变朝向过渡态。(C)2003 Elsevier Ltd.保留所有权利。
N-Acetyl-L-glutamate kinase (NAGK), the structural paradigm of the enzymes of the amino acid kinase family, catalyzes the phosphorylation of the gamma-COO- group of N-acetyl-L-glutamate (NAG) by ATP We determine here the crystal structures of NAGK complexes with MgADP, NAG and the transition-state analog AlF4-; with MgADP and NAG; and with ADP and SO42-. Comparison of these structures with that of the MgAMPPNP-NAG complex allows to delineate three successive steps during phosphoryl transfer: at the beginning, when the attacking and leaving O atoms and the P atom are imperfectly aligned and the distance between the attacking O atom and the P atom is 2.8 Angstrom; midway, at the bipyramidal intermediate, with nearly perfect alignment and a distance of 2.3 Angstrom; and, when the transfer is completed. The transfer occurs in line and is strongly associative, with Lys8 and Lys217 stabilizing the transition state and the leaving group, respectively, and with Lys61, in contrast with an earlier proposal, not being involved. Three water molecules found in all the complexes play, together with Asp162 and the Mg, crucial structural roles. Two glycine-rich loops (beta1-alphaA and beta2-alphaB) are also very important, moving in the different complexes in concert with the ligands, to which they are hydrogen-bonded, either locking them in place for reaction or stabilizing the transition state. The active site is too narrow to accommodate the substrates without compressing the reacting groups, and this compressive strain appears a crucial component of the catalytic mechanism of NAGK, and possibly of other enzymes of the amino acid kinase family such as carbamate kinase. Initial binding of the two substrates would require a different enzyme conformation with a wider active site, and the energy of substrate binding would be used to change the conformation of the active center, causing substrate strain towards the transition state. (C) 2003 Elsevier Ltd. All rights reserved.