Analysis of the structural determinants underlying discrimination between substrate and solvent in beta-phosphoglucomutase catalysis.
Analysis of the structural determinants underlying discrimination between substrate and solvent in beta-phosphoglucomutase catalysis.
复制标题
DOI：
10.1021/bi801653r
复制
发表时间：
2009-03-10
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano, Debra
中科院分区：
文献类型：
--
作者：
Dai, Jianying;Finci, Lorenzo;Zhang, Chunchun;Lahiri, Sushmita;Zhang, Guofeng;Peisach, Ezra;Allen, Karen N.;Dunaway-Mariano, Debra
卤酸脱卤酶超家族（HADSF）中的β-磷酸葡萄糖糖糖化酶（β-PGM）利用核心结构域活性位点Asp8介导β-葡萄糖1,6-（双）磷酸（βG1,6bisP）向βG1P的磷酸化，催化β-葡萄糖1-磷酸（βG1P）向葡萄糖6-磷酸（G6P）的转化。在此，我们探索了β-PGM phospho-Asp8在活性位点必须保持对溶剂开放以允许结合产物G6P与底物βG1P交换的过程中避免水解的机制。基于结构信息，提出了一种催化模型，其中一般酸/碱（Asp10）侧链从与结构域连接器Thr16-Ala17形成氢键的位置移动到与底物离开基O和帽结构域His20-Lys76对形成氢键的功能位置。核心结构域活性位点内一般酸/碱的重新定位与底物诱导的帽结构域在核心结构域上的闭合是协调的。该模型预测，Asp10是一般酸/碱催化和酶在帽闭构象中稳定所必需的。它还预测铰链残基Thr16在产生结构域-结构域结合中起关键作用，在帽打开构象中需要与Thr16骨架酰胺NH的氢键相互作用来防止磷酸- asp8水解，并且His20-Lys76对在底物诱导的帽闭合中起重要作用。该模型通过Asp10、Thr16、His20和Lys76位点突变体的动力学分析进行了检验。用Ala、Ser、Cys、Asn或Glu代替Asp10没有观察到活性。用Pro取代连接体残基Thr16的动力学结果包括：Asp8被βG1、6bisP磷酸化的速率降低，将βG1P转化为G6P的磷酸化酶的循环速率降低，以及磷酸化的Asp8向水转移的速率提高。2.7 Å分辨率下的T16P突变体的x射线结构提供了该酶在非自然帽打开构象中的快照，其中Asp10侧链位于核心结构域活性位点。His20和Lys76位点突变体对asp8介导的βG1、6bisP和βG1P之间磷酸化转移的催化活性降低，但磷酸化- asp8的水解速率没有降低。综上所述，结果支持底物诱导的催化模型，其中βG1P与核心结构域的结合促进了通用酸/碱Asp10的招募到催化位点并诱导帽盖闭合。
The β-phosphoglucomutase (β-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of β-glucose 1-phosphate (βG1P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active-site to mediate phosphoryl transfer from β-glucose 1,6-(bis)phosphate (βG1,6bisP) to βG1P. Herein we explore the mechanism by which hydrolysis of the β-PGM phospho-Asp8 is avoided during the time that the active site must remain open to solvent in order to allow the exchange of the bound product G6P with the substrate βG1P. Based on structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-Ala17 of the domain-domain linker, to a functional position where it forms a hydrogen bond to the substrate leaving-group O and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that Asp10 is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain-domain association, that hydrogen bond interaction with the Thr16 backbone amide NH is required to prevent phospho-Asp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thr16, His20, and Lys76 site-directed mutants. Replacement of the Asp10 by Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement of linker residue Thr16 with Pro include a reduced rate of Asp8 phosphorylation by βG1,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert βG1P to G6P, and an enhanced rate of phosphoryl transfer from phospho-Asp8 to water. The X-ray structure of the T16P mutant at 2.7 Å resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core-domain active site. The His20 and Lys76 site-directed mutants show reduced activity in catalysis of the Asp8-mediated phosphoryl transfer between βG1,6bisP and βG1P but no reduction in the rate of phospho-Asp8 hydrolysis. Taken together, the results support a substrate induced-fit model of catalysis in which βG1P binding to the core domain facilitates recruitment of the general-acid/base Asp10 to the catalytic site and induces cap closure.
登录
查看更多内容
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1046/j.1432-1327.1999.00373.x
发表时间：
1999-05-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Jolly, L;Ferrari, P;Mengin-Lecreulx, D
通讯作者：
Mengin-Lecreulx, D
DOI：
10.1107/s0907444902016657
发表时间：
2002-11-01
影响因子：
2.2
作者：
Adams, PD;Grosse-Kunstleve, RW;Terwilliger, TC
通讯作者：
Terwilliger, TC
影响因子：
2.9
作者：
Lahiri, SD;Zhang, GF;Allen, KN
通讯作者：
Allen, KN
影响因子：
56.9
作者：
Lahiri, SD;Zhang, GF;Allen, KN
通讯作者：
Allen, KN