Understanding Extended Active Sites in Enzymes

生物化学中的一个基本问题是了解酶是如何工作的。该项目旨在更深入地了解自然界如何设计酶活性位点，特别是不与反应底物分子直接接触的残基。结构和生物化学研究现在已经表征了数百种酶的活性位点，并且几乎所有这些研究都集中在与反应底物直接接触的氨基酸上;这些残基可以被视为活性位点的第一层。最近的理论预测和实验研究强烈表明，活性位点第一层外的残基对催化作用也非常重要。本计画的目标是将联合收割机理论、计算与实验结合起来，以建立酶中空间延伸活性位的普遍性，以显示此现象是可预测的计算，并阐明空间上遥远的残基参与酶催化的机制。一个这样的机制，和一个强烈的当前感兴趣的问题，是蛋白质动力学在催化过程中的作用。之所以选择这里研究的例子，是因为它们有不同类型的有趣的动力学过程在起作用。在DNA聚合酶III中，聚合酶亚基的运动调节DNA结合，并可能负责检查碱基对形成的保真度。对于鸟氨酸转氨甲酰酶，据报道，第一反应物分子的结合会发生诱导的适合构象变化。对于甘氨酰胺核糖核苷酸转化酶，已经提出了一种pH依赖性分子开关，用于激活催化位点的螺旋到螺旋的转变。初步证据表明，第一层壳外的残留物在这些过程中发挥着核心作用。对于这三种酶，突变将在计算预测对催化重要的第二和第三壳中的位置处进行，并且这些突变体将在动力学和结构上表征。将对野生型和变体进行分子动力学（MD）模拟，以确定突变是否影响蛋白质的动力学，以及这种运动是否有助于催化，预测将使用广角X射线溶液（WAXS）散射进行实验测试。远程残基参与酶催化的原则的建立，并证明这种参与是可预测的计算，将是非常有用的酶工程和机制的研究。为该项目开发的计算方法将通过网络免费提供给科学界，用于研究，包括蛋白质工程和酶机制研究，以及商业应用。这个项目将提供更好地了解自然界的酶活性位点的设计和酶如何影响催化作用。这种更好的理解有助于开发新技术，如更清洁的“绿色”工业工艺、可持续的环境补救方法和酶促生物燃料合成。本项目所述的高素质科学家的培训对区域高科技经济和美国在全球经济中的竞争力至关重要。两名博士生将接受计算方法，蛋白质表达，突变，纯化，动力学和结合分析，晶体结构测定和其他X射线散射方法的培训。该项目的部分内容将被整合到分子建模课程中，学生积极参与一些计算和建模工作。本科生的研究参与将继续下去。 该项目将继续与主要本科院校（PUI）的教师进行合作，包括西班牙裔服务PUI。计算和其他研究工具的实践演示将继续在PUI和内城K-12学生。项目团队成员将继续广泛参与美洲原住民学生，专业人士和社区团体。

A fundamental problem in biochemistry is to understand how enzymes work. This project seeks greater insight into how nature designs enzyme active sites, particularly the residues that are not in direct contact with the reacting substrate molecule(s). Structural and biochemical studies have now characterized the active sites of hundreds of enzymes, and nearly all of these studies have focused on the amino acids in direct contact with the reacting substrate; these residues may be regarded as the first layer of the active site. Recent theoretical predictions and experimental studies strongly suggest that residues outside the first layer of the active site can also be very important for catalysis. The goals of this project are to combine theory, computation, and experiment to establish the prevalence of spatially extended active sites in enzymes, to show that this phenomenon is predictable computationally, and to elucidate the mechanisms by which spatially remote residues participate in enzyme catalysis. One such mechanism, and a problem of intense current interest, is the role of protein dynamics in the catalytic process. The examples studied here have been chosen because they have different kinds of interesting dynamical processes in play. In DNA polymerase III, motions in the polymerase subunit modulate DNA binding and are likely responsible for checking the fidelity of base-pair formation. For ornithine transcarbamylase, an induced fit conformational change has been reported to occur with the binding of the first reactant molecule. For glycinamide ribonucleotide transformylase, a pH-dependent molecular switch has been proposed for a coil-to-helix transition that activates the catalytic site. Preliminary evidence suggests that residues outside the first shell play central roles in each of these processes. For these three enzymes, mutations will be made at positions in the second and third shells that are predicted computationally to be important for catalysis, and these mutants will be characterized kinetically and structurally. Molecular dynamics (MD) simulations will be performed on the wild type and variants to determine whether the mutations affect the dynamics of the protein, and whether this motion contributes to catalysis, predictions that will be tested experimentally using wide-angle x-ray solution (WAXS) scattering. The establishment of principles governing remote residue participation in enzyme catalysis, and evidence that such participation is predictable computationally, will be very useful for enzyme engineering and mechanistic studies. The computational methods developed for this project will be made freely available to the scientific community via the web for use in research, including protein engineering and enzyme mechanism studies, and in commercial applications. This project will provide better understanding of nature's design of enzyme active sites and of how enzymes affect catalysis. Such improved understanding can help in the development of novel technologies, such as cleaner, "green" industrial processes, sustainable methods for environmental remediation, and enzymatic biofuel synthesis. The training of highly qualified scientists as described in this project is vital to the regional high-tech economy and to U.S. competitiveness in the global economy. Two doctoral students will be trained in computational methods, in protein expression, mutation, purification, kinetics and binding assays, crystal structure determination, and other x-ray scattering methods. Parts of the project will be integrated into the Molecular Modeling course, with active student participation in some of the computational and modeling work. Research participation by undergraduate students will continue. This project will continue current collaborations with faculty members at primarily undergraduate institutions (PUIs), including a Hispanic-serving PUI. Hands-on demonstrations of computational and other research tools will continue at PUIs and to inner city K-12 students. Extensive participation by project team members in Native American student, professional, and community groups will continue.