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Characterization of the dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex.

机译：大肠杆菌丙酮酸脱氢酶多酶复合物E1组分动力学的表征。

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摘要

The E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex (EC 1.2.4.1) is a thiamin diphosphate dependent enzyme that catalyzes oxidative decarboxylation of pyruvate. In the E1 component, two active center loops (an inner loop formed by residues 401-413, and outer loop formed by residues 541-557) become organized only on binding a intermediate analog which is capable of forming a stable thiamin diphosphate-bound covalent intermediate. In the ordered conformation many of the inner loop residues come into proximity of the active center and form a part of the active site channel. Kinetic, spectroscopic, and crystallographic studies on some inner loop residues and their variants led to the conclusion that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for (1) predecarboxylation events, (2) for sequestering active site chemistry from undesirable side reactions, (3) entry of ThDP and second molecule substrate and (4) for promoting communication between the E1 and E2 components of E. coli pyruvate dehydrogenase multienzyme complex.;Studies using site specific labeling (SSL) with thiol-directed probes sensitive to their local environment has immense potential to characterize the dynamics of these important regions. For this purpose five of six cysteine residues in parental E1 (120, 259, 575, 610, 654 and 770) were converted to alanines, the sixth C259 to asparagine, by site directed mutagenesis. This E1 variant without cysteines showed ∼4-fold reduction in activity compared to parental E1 in component-specific as well as overall complex activity assays. Moreover, reintroduction of cysteine for SSL studies in regions 1-55 (I11C) and 401-413 (Q408C) did not result in significant reduction in E1 activity retaining ∼40% and 80% overall activity, respectively, compared to the cysteineless E1 variant and ThDP or pre-decarboxylation steps. These results affirm that a cysteine-free construct creates a reliable system for characterization of E1 loop dynamics with the help of SSL studies.;Protein motions are ubiquitous and are intrinsically coupled to catalysis; their specific roles, however, remain largely elusive. Initial studies indicated that dynamic loops at the active center of the E1 component are essential for many of its catalytic functions starting from predecarboxylation steps to the transfer of the acetyl moiety to the E2 component. The kinetic resolution of pre-decarboxylation steps afforded by time-resolved circular dichroism studies revealed that formation of enzyme-ThDP-pyruvate Michaelis complex is extremely fast and could be diffusion limited. However, covalent addition of substrate to enzyme bound coenzyme (C-C bond formation) is found to be rate determining not only for pre-decaboxylation steps but also for overall E1 catalysis. Monitoring the steady state and time resolved kinetics of E1 and its loop variants at various solution viscosities revealed that kinetic steps, particularly C-C covalent bond formation, are indeed modulated by the loop dynamics. These observations taken together were strongly suggestive of an intimate correlation of catalysis and loop dynamics.;To determine the qualitative nature of these important and potentially correlated motions we used EPR spectroscopy, a highly sensitive method demonstrated in many studies to be exquisitely sensitive reporter of conformational changes. A cysteine-free E1 construct created for site-specific labeling with nitroxide on the inner loop revealed ligand induced conformational dynamics of the loop and a slow ‘open-close’ conformational equilibrium in the unliganded state. A 19F NMR label placed at various positions on the inner loop revealed motion on the ms to s time scale and led to a quantitative correlation of E1 catalysis and loop dynamics for the 200,000 Da protein. Thermodynamic studies showed that these motions coupled to catalysis reduce free energy of activation of covalent addition of substrate to the enzyme bound thiamin diphosphate by influencing both enthalpic and entropic components. This observation is significant considering the vast experimental data supporting electrostatic catalysis and differences surrounding the ‘dynamical hypothesis’ of enzyme catalysis. While data presented in this study do not argue against a generalized view favoring electrostatic catalysis in enzymes, they do place limitations on its predominance in E1. At the same time, the data strongly indicate ‘energetic harvesting of dynamics’ also play important role in catalysis as has been proposed by recent experimental and theoretical studies.;These results demonstrate efficient coupling of catalysis and regulation with enzyme dynamics and more importantly suggests a mechanism by which it may be achieved in a key branch-point enzyme in sugar metabolism reinforcing the hypothesis ascribing catalytic and regulatory roles to enzyme dynamics.
机译：大肠杆菌丙酮酸脱氢酶多酶复合物（EC 1.2.4.1）的E1成分是硫胺素二磷酸依赖性酶，可催化丙酮酸的氧化脱羧。在E1组分中，两个活性中心环（由残基401-413形成的内环和由残基541-557形成的外环）仅在结合能够形成稳定的硫胺二磷酸结合的共价键的中间体类似物上时才组织起来中间。在有序构象中，许多内环残基进入活性中心附近并形成活性位点通道的一部分。对一些内环残基及其变体的动力学，光谱和晶体学研究得出的结论是，His-407侧翼的带电残基对于内环的稳定/有序化很重要，从而有助于完成活性位点。结果还表明，动态内环的有序到无序的混乱对于（1）预脱羧事件，（2）从不希望的副反应中隔离活性位点化学反应，（3）ThDP和第二分子底物的进入以及（4）至关重要。 ），以促进大肠杆菌丙酮酸脱氢酶多酶复合物的E1和E2组分之间的交流。;使用位点特异性标记（SSL）和对本地环境敏感的硫醇定向探针进行的研究具有表征这些重要区域动态的巨大潜力。为此，通过定点诱变将亲本E1中六个半胱氨酸残基中的五个（120、259、575、610、654和770）转化为丙氨酸，第六个C259转化为天冬酰胺。在组分特异性以及总体复杂活性测定中，这种不具有半胱氨酸的E1变体的活性比亲本E1降低了约4倍。此外，与无半胱氨酸的E1变体相比，在1-55（I11C）和401-413（Q408C）的SSL研究中重新引入半胱氨酸不会导致E1活性显着降低，分别保持约40％和80％的总体活性。 ThDP或预脱羧步骤。这些结果肯定了无半胱氨酸的构建体通过SSL研究创建了一个表征E1环动力学的可靠系统。蛋白质运动无处不在，并固有地与催化作用相关；然而，它们的具体作用仍然难以捉摸。初步研究表明，E1组分活性中心的动态环对于从预脱羧步骤到乙酰基部分转移到E2组分开始的许多催化功能都是必不可少的。时间分辨的圆二色性研究提供的预脱羧步骤的动力学拆分显示，酶-ThDP-丙酮酸米氏菌复合物的形成非常快，并且可能受到扩散限制。然而，发现底物共价添加到酶结合的辅酶上（C-C键形成）不仅决定了脱十aboxylation步骤的速率，而且决定了整个E1催化速率。在各种溶液粘度下监测E1及其环变体的稳态和时间分辨动力学，发现动力学步骤（尤其是C-C共价键形成）确实受到环动力学的调节。这些观察结果加在一起，强烈暗示了催化作用和环动力学的密切相关。为了确定这些重要且可能相关的运动的定性性质，我们使用了EPR光谱技术，该方法在许多研究中均被证明是非常敏感的构象报告物。变化。在内环上用氮氧化物进行位点特异性标记的无半胱氨酸E1构建体显示出配体诱导的环构象动力学，以及在未配体状态下缓慢的“开闭”构象平衡。放置在内环各个位置的19F NMR标记揭示了毫秒到s时标的运动，并导致了200,000 Da蛋白的E1催化和环动力学的定量相关。热力学研究表明，这些运动与催化作用相关联，通过影响焓和熵的成分，降低了底物共价添加到酶结合的硫胺素二磷酸中的活化自由能。考虑到支持静电催化的大量实验数据以及酶催化“动力学假设”周围的差异，这一发现意义重大。尽管本研究中提供的数据并未与赞成酶中静电催化的普遍观点相抵触，但它们确实限制了其在E1中的优势。与此同时，这些数据强烈表明“动力学的能量收集”在催化作用中也起着重要作用，正如最近的实验和理论研究所提出的那样。这些结果证明了催化和调节与酶动力学的有效耦合，更重要的是提出了一种机理，通过这种途径糖代谢中的关键分支点酶可能会实现这一点，从而加强了假设，因为酶动力学具有催化和调节作用。

著录项

  • 作者

    Kale, Sachin.;

  • 作者单位

    Rutgers The State University of New Jersey - Newark.;

  • 授予单位 Rutgers The State University of New Jersey - Newark.;
  • 学科 Chemistry Biochemistry.;Biophysics General.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 201 p.
  • 总页数 201
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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