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首页> 外文期刊>Biochemistry >Protein Interactions in the Human Methionine Synthase-Methionine Synthase Reductase Complex and Implications for the Mechanism of Enzyme Reactivation
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Protein Interactions in the Human Methionine Synthase-Methionine Synthase Reductase Complex and Implications for the Mechanism of Enzyme Reactivation

机译：人甲硫氨酸合酶 - 蛋氨酸合酶还原酶复合酶复合蛋白相互作用及其对酶再激活机制的影响

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摘要

Methionine synthase (MS) is a cobalamin-dependent enzyme.It transfers a methyl group from rnethyltetrahydrofolate to homocysteine forming methionine and tetrahydrofolate.On the basis of sequence similarity with Escherichia coli cobalamin-dependent MS (MetH),human MS comprises four discrete functional modules that bind from the N- to C-terminus,respectively,homocysteine,rnethyltetrahydrofolate,cobalamin,and S-adenosylmethionine (AdoMet).The C-terminal activation domain also interacts with methionine synthase reductase (MSR),a NADPH-dependent diflavin oxidoreductase required for the reductive regeneration of catalytically inert cob(II)alamin (which is formed every 200-1000 catalytic cycles of MS) to cob(I)alamin.We have investigated complex formation between the (i) MS activation domain and MSR and (ii) MS activation domain and the isolated FMN-binding domain of MSR.We show that the MS activation domain interacts directly with the FMN-binding domain of MSR.Binding is weakened at high ionic strength,emphasizing the importance of electrostatic interactions at the protein-protein interface.Mutagenesis of conserved lysine residues (Lys1071 and Lys987) in the human activation domain weakens this protein interaction.Chemical cross-linking demonstrates complex formation mediated by acidic residues (FMN-binding domain) and basic residues (activation domain).The activation domain and isolated FMN-domain form a 1:1 complex,but a 1:2 complex is formed with activation domain and MSR.The midpoint reduction potentials of the FAD and FMN cofactors of MSR are not perturbed significantly on forming this complex,implying that electron transfer to cob(n)alamin is endergonic.The kinetics of electron transfer in MSR and the MSR-activation domain complex are similar.Our studies indicate (i) conserved binding determinants,but differences in protein stoichiometry,between human MS and bacterial MetH in complex formation with redox partners;(ii) a substantial endergonic barrier to electron transfer in the reactivation complex;and (iii) a lack of control on the thermodynamics and kinetics of electron transfer in MSR exerted by complex formation with activation domain.The structural and functional consequences of complex formation are discussed in light of the known crystal structure of human activation domain and the inferred conformational heterogeneity of the multidomain MSR-MS complex.
机译：甲硫氨酸合成酶（MS）是一种钴胺依赖性酶。将甲基来自丙基甲基四氢醇酸酯转移到同型半胱氨酸形成甲硫氨酸和四氢溶胶的基础上。序列相似性与大肠杆菌钴钴蛋白依赖性MS（甲基），人体MS包括四个离散函数模块从N-至C-末端结合，分别是同型半胱氨酸，rnethylahtahyrofolate，钴胺素和S-腺苷甲硫氨酸（Adomet）。C-末端活化结构域也与甲硫氨酸合酶还原酶（MSR）相互作用，所需的NADPH依赖性Diflavin氧化酶。对于催化惰性COB（II）的还原再生酰胺（每200-1000个催化循环的MS）到COB（I）阿拉胺酰胺。我们在（I）MS激活结构域和MSR之间研究了复杂的形成和（II） ）MSR.WE的MS激活结构域和分离的FMN结合域显示MS激活结构域与MSR的FMN结合结构域直接相互作用。绕线在高离子下削弱IC强度，强调蛋白质蛋白质界面处的静电相互作用的重要性。在人活化结构域中的保守赖氨酸残基（Lys1071和Lys987）的静电相互作用削弱该蛋白质相互作用。化学交联显示通过酸性残基介导的复杂形成（FMN-结合域）和基本残留物（激活域）。活化结构域和分离的FMN-结构域形式1：1复合物，但是用活化域和MSR形成1：2复合物。FAD和FMN辅因子的中点减少电位MSR在形成该复合物上没有明显扰动，这意味着电子转移到COB（N）Alamin是idergeronic。MSR和MSR激活域复合物中的电子转移动力学是相似的。我们的研究表明（i）保守的结合决定簇但是蛋白质化学计量的差异，用氧化还原合作伙伴在络合物形成的人MS和细菌甲基中的差异;（ii）对电子TRA的实质性抗结构屏障在重新激活复合物中Nsfer;（iii）通过用活化结构域的复杂形成施加的MSR中电子转移的热力学和动力学对缺乏控制。根据已知的晶体结构讨论复杂形成的结构和功能后果人体激活结构域和多麦田MSR-MS复合物的推断构象异质性。

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  • 来源
    《Biochemistry》 |2007年第23期|共14页
  • 作者

    Kirsten R.Wolthers; Nigel S.Scrutton;

  • 作者单位

    Faculty of Life Sciences Manchester Interdisciplinary Biocentre University of Manchester 131 Princess Street Manchester Ml 7DN United Kingdom;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
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