Single Molecule Enzymology

对给定酶的单个分子进行的测量表明，不同的单个分子具有不同的性质。这种现象被称为酶的异质性。几种不同的实验方法已被用于几种不同的酶。在所有情况下均观察到异质性。报道的非均相性质包括催化速率、催化活化能和电泳迁移率。申请人实验室研究的长期目标是了解这种异质性的原因和生物学作用。申请人实验室中的大多数工作是使用β-半乳糖苷酶或碱性磷酸酶作为模型酶进行的，尽管最近这项工作已经扩展到酶β-葡糖醛酸糖苷酶。这些测定使用基于毛细管电泳的方法进行。 这份提案由三个短期目标组成，这些目标符合我们的长期目标。本研究的第一个短期目标是扩大我们的酶分子库，可以在单分子水平上使用毛细管电泳进行测定。最初的工作将集中在改进和扩大我们的β-葡萄糖醛酸酶的测定和开发一种过氧化物酶和蛋白酶的测定。 此外，我们打算开发一种用于分离和检测代谢物的方法，这将是有用的单细胞代谢组学。 第二个目标是使用牛碱性磷酸酶（一种糖基化酶）作为模型酶，确定翻译后糖基化对酶异质性的影响。最初，糖基化状态将使用靶向不同糖部分的非变性去糖基化试剂盒改变，并测定样品以确定对催化速率、电泳迁移率和催化活化能分布的影响。然后将牛酶在巴斯德毕赤酵母中表达，并进行诱变以确定选择性去除糖基化位点的效果。 自由区毛细管电泳是一种常用的蛋白质分离技术。我们的研究已经表明，酶分子本质上是异构的。这代表了使用自由区毛细管电泳分离蛋白质的基本限制。该提案的第三个短期目标是确定导致电泳异质性的因素，以预测哪种类型的蛋白质结构可以以高分辨率分离，以及哪种类型可能显示使用该技术的较低分辨率。

Measurements made on individual molecules of a given enzyme have shown that the different individual molecules have different properties. The phenomenon has been termed enzyme heterogeneity'. Several different experimental approaches have been used on several different enzymes. In all cases heterogeneity has been observed. Reported heterogeneous properties include catalytic rate, activation energy of catalysis and electrophoretic mobility. The long term goal of the research in the applicant's laboratory is to understand the cause and biological role of this heterogeneity. Most work in the applicant's laboratory has been done using beta-galactosidase or alkaline phosphatase as model enzymes, although recently this work has been expanded to the enzyme beta-glucuronidase. These assays are performed using a capillary electrophoresis-based approach. This proposal is comprised of three short term goals that fit into our longer term goal. The first short term objective of this study is to expand our repertoire of enzyme molecules which can be assayed at the single molecule level using capillary electrophoresis. Initial work will focus on improving and expanding our assay for beta-glucuronidase and developing an assay for peroxidase and proteases. Additionally, we intend to develop a method for the separation and detection of metabolites that will be useful in single cell metabolomics. The second goal is to determine the effect of post-translational glycosylation on enzyme heterogeneity using bovine alkaline phosphatase, which is a glycosylated enzyme, as the model enzyme. Initially glycosylation states will be altered using non-denaturation deglycosylation kits targeting different sugar moieties and samples assayed to determine the effect on the distributions of catalytic rates, electrophoretic mobility and activation energy of catalysis. The bovine enzyme will then be expressed in P. pastoris and mutagenesis be performed to determine the effect of selective removal of glycosylation sites. Free zone capillary electrophoresis is a commonly used technique for analytical protein separations. Our research has shown that enzyme molecules are inherently electrophoretically heterogeneous. This represents a fundamental limit to resolution in protein separations using free zone capillary electrophoresis. The third short term goal of this proposal is to determine the factors that cause electrophoretic heterogeneity in order to predict which type of protein structures can be separated with high resolution and which types may show lower resolution using this technique.