移动环的构象调节控制组氨酸合成的 (βα)8 管状酶 HisF 的催化作用

IF 8.5 Q1 CHEMISTRY, MULTIDISCIPLINARY
Enrico Hupfeld, Sandra Schlee, Jan Philip Wurm, Chitra Rajendran, Dariia Yehorova, Eva Vos, Dinesh Ravindra Raju, Shina Caroline Lynn Kamerlin*, Remco Sprangers* and Reinhard Sterner*, 
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引用次数: 0

摘要

环路运动对酶活性的整体意义已被普遍接受。然而,这种运动是否以及如何控制催化的不同步骤,在很大程度上仍不清楚。我们以(βα)8-桶酶 HisF(咪唑甘油磷酸合成酶的环化酶亚基)的移动活性位点β1α1-环(loop1)为例研究了这一问题。含有单个保守氨基酸突变的 Loop1 变体在底物 N′-[(5′-磷酰)甲酰亚胺基]-5-氨基咪唑-4-甲酰胺核糖核苷酸(PrFAR)和氨转化为产物咪唑甘油磷酸酯(ImGP)和 5-氨基咪唑-4-甲酰胺核糖核苷酸(AICAR)的过程中显示出急剧下降的速率。一项包括停流动力学、X 射线晶体学、核磁共振光谱学和分子动力学模拟在内的综合机理分析检测出了 loop1 的三种构象(开放、分离和封闭),野生型 HisF 和受功能影响的 loop1 变体在这些构象的数量上存在差异。瞬时停流动力学实验表明,wt-HisF 通过诱导-拟合机制与 PrFAR 结合,而催化受损的 loop1 变体则通过简单的双态机制与 PrFAR 结合。我们的研究结果表明,PrFAR 诱导形成的 loop1 闭合构象使活性位点残基处于化学周转的有效取向,我们证明这是 HisF 催化的限速步骤。环化酶反应后,闭合环构象不稳定,这有利于形成分离和开放构象,从而促进产物 ImGP 和 AICAR 的释放。我们的数据证明了活性位点环的不同构象如何有助于不同的催化步骤,这一发现可能对(βα)8-桶酶及其他酶的反应机制具有广泛的意义。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα)8-Barrel Enzyme of Histidine Biosynthesis HisF

Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα)8-Barrel Enzyme of Histidine Biosynthesis HisF

The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N′-[(5′-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (βα)8-barrel enzymes and beyond.

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CiteScore
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