The Enzymatic Pathway of a Ubiquitin-Like Modification

泛素样修饰类似于其他大分子化学，如 转录和DNA修复，因为它们需要多个步骤，这些步骤由多个 蛋白质复合体。更好地理解多蛋白质机制如何催化泛素- Like修改从根本上很重要，并将显著提高我们对 多蛋白质复合体进行大分子化学。 这项提案的总体目标是研究蛋白质修饰的酶学。 通过类似泛素的小修饰物(相扑)。相扑，它被确立为一种重要的 翻译后修饰，是一个很好的模型系统的酶研究，并已 被提议作为癌症治疗的靶点，因为它的酶水平在 癌症。关于酶学的一个谜团是，结合底物距离太远 晶体结构中的催化位置来解释催化作用，流行的观点是 必须发生构象变化才能使它们更接近。正如我们的 初步研究部分，虽然可用的结构是稳定的络合物，但稳定性较差 尚未确定的相互作用对酶循环至关重要。我们会 研究较弱的相互作用如何引导底物移位到催化活性 并在酶中诱导变构效应。我们将在结构上使用核磁共振波谱 研究，这特别适合于表征弱蛋白质复合体。互补性 生化和酶动力学分析将被用来确定每个相互作用在 酶机制。拟议的研究可能会导致关于 酶催化泛素样修饰，并提高我们对主题和 多蛋白质机制如何催化大分子反应的变化。

Ubiquitin-like modifications are similar to other macromolecular chemistry, such as transcription and DMArepair, in that they require multiple steps that are carried out by multi- protein complexes. Better understanding of how the multi-protein machinery catalyzes ubiquitin- like modifications is fundamentally important and will significantly improve our knowledge of how multi-protein complexes carry out macromolecular chemistry. The overall goal of this proposal is to investigate the enzymology of protein modifications by the small ubiquitin-like modifier (SUMO). Sumoylation, which is established as an important post-translational modification, is a good model system for enzymology studies, and has been proposed as a cancer therapeutic target, because of the increased levels of its enzymes in cancers. A mystery about the enzymology is that the binding substrates are too far apart from the catalytic sites in crystal structures to account for catalysis, and the prevailing view is that conformational changes must take place to bring them into close proximity. As shown in our Preliminary Studies section, while the available structures are stable complexes, less stable interactions that have not been characterized are critical to the enzymatic cycle. We will investigate how the weaker interactions may guide substrate translocations to catalytic active sites, and induce allosteric effects in the enzymes. We will use NMR spectroscopy in structural studies, which is especially suitable for characterizing weak protein complexes. Complementary biochemical and enzyme kinetic analysis will be used to identify the role of each interaction in the enzyme mechanism. The proposed studies will likely result in a paradigm shift regarding the enzymes catalyzing ubiquitin-like modifications, and improve our knowledge of the themes and variations of how multi-protein machineries catalyze macromolecular reactions.