Molecular Mechanisms of Dietary Fat Digestion by Pancreatic Lipases

项目摘要 我们的长期目标是阐明胰脂肪酶消化膳食脂肪的分子机制。 在本申请中，我们将集中在影响底物特异性的脂解步骤。脂肪酶不同于 在脂肪酶中作用于水溶性底物酯酶必须吸收到底物的界面 在底物结合到活性位点之前，此外，胰腺脂肪酶的原型，胰腺 甘油三酯脂肪酶（PTL）需要辅脂酶吸收到界面。重要的是，吸收中的突变 辅脂酶的表面可以改变PTL-辅脂酶复合物的底物偏好，从而强调了 吸收在确定底物特异性中的重要性。因此，三个不同的步骤--辅脂酶结合， 界面吸收和活性部位的底物结合--都影响底物特异性。探讨 介导PTL与底物相互作用的分子元件，我们将利用一个紧密的 PTL同源物，胰脂肪酶相关蛋白2（PLRP 2）。这些脂肪酶具有高度保守的初级 结构，共享一个共同的三维结构和共享共同的催化机制;然而，它们 具有完全不同的酶特性。PTL优选甘油三酯，而PLRP 2也切割半乳糖脂 和磷脂。此外，PTL明显需要辅脂酶，而PLRP 2需要辅脂酶 仍然存在争议。我们的中心假设是，特定的结构域控制着蛋白质合成的每一步。 胰脂肪酶和底物之间的相互作用。要识别在确定 PTL和PLRP 2的底物特异性，我们将利用结构和动力学性质的可用信息 这些脂肪酶。我们的这种方法基于我们已发表的和初步的数据的多个观察。第一章 结构研究和我们发表的研究PTL-辅脂酶复合物指导了鉴定 影响辅脂酶与PTL结合的不同结构域。2)分析结构数据和我们的初步 研究已经鉴定了PTL-辅脂酶和PLRP 2中影响这些酶的吸收的关键残基， 脂肪酶到脂肪乳剂。3)PTL和PLRP 2的结构比较及初步分析 鉴定了可以介导底物特异性差异的结构域。实现我们的具体目标 将提供胰脂肪酶与底物相互作用中每一步的分子细节。最后， 我们将能够合理地设计脂肪酶的酶性质，以满足特定的治疗或 工业需要。

Project Summary Our long-term goal is to elucidate the molecular mechanisms of dietary fat digestion by pancreatic lipases. In this application, we will focus on the steps in lipolysis that influence substrate specificity. Lipases differ from esterases that act on water soluble substrates in that lipases must absorb to the interface of the substrate emulsion before substrate binds in the active site. In addition, the archetype of pancreatic lipases, pancreatic triglyceride lipase (PTL), requires colipase to absorb to interfaces. Importantly, mutations in the absorption surface of colipase can change the substrate preference of the PTL-colipase complex underscoring the importance of absorption in determining substrate specificity. Thus, three distinct steps--colipase binding, interfacial absorption and substrate binding in the active site--all influence substrate specificity. To investigate the molecular elements that mediate the interaction of PTL with substrate, we will take advantage of a close PTL homologue, pancreatic lipase related protein 2 (PLRP2). These lipases have highly conserved primary structures, share a common three-dimensional structure and share common catalytic machinery; yet, they have quite different enzymatic properties. PTL prefers triglycerides whereas PLRP2 also cleaves galactolipids and phospholipids. Furthermore, PTL clearly requires colipase whereas the PLRP2 requirement for colipase remains controversial. Our central hypothesis is that specific structural domains govern each step of the interaction between pancreatic lipases and substrate. To identify domains involved in determining the substrate specificity of PTL and PLRP2, we will utilize available information on structure and kinetic properties of these lipases. We base this approach on multiple observations from our published and preliminary data. 1) Structural studies and our published studies of the PTL-colipase complex have guided the identification of distinct domains that influence colipase binding to PTL. 2) Analysis of structural data and our preliminary studies have identified critical residues in PTL-colipase and PLRP2 that influence the absorption of these lipases to lipid emulsions. 3) Comparison and preliminary analysis of the structures of PTL and PLRP2 identified domains that can mediate the differences in substrate specificity. Completion of our Specific Aims will provide molecular details about each step in the interaction of pancreatic lipases with substrate. Ultimately, we will be able to rationally engineer the enzymatic properties of lipases to meet specific therapeutic or industrial needs.