Molecular Mechanisms of Dietary Fat Digestion by Pancreatic Lipases

项目摘要 我们的长期目标是阐明胰腺脂肪酶消化膳食脂肪的分子机制。 在本应用中，我们将重点介绍影响底物特异性的脂解步骤。脂肪酶不同于 作用于水溶性底物的酯酶必须吸收到底物的界面上 底物结合在活性部位之前的乳状液。此外，胰腺脂肪酶的原型，胰腺 甘油三酯脂肪酶(PTL)，需要脂肪酶吸收到界面上。重要的是，吸收过程中的突变 脂酶的表面可以改变PTL-脂酶复合体的底物偏好，强调了 吸收在确定底物专一性中的重要性。因此，有三个不同的步骤--胶脂酶结合， 活性部位的界面吸收和底物结合都会影响底物的专一性。去调查 调节PTL与底物相互作用的分子元素，我们将利用 PTL同源物，胰腺脂肪酶相关蛋白2(PLRP2)。这些脂肪酶具有高度保守的初级 结构，共享共同的三维结构并共享共同的催化机械；然而，它们 具有完全不同的酶性质。PTL偏爱甘油三酯，而PLRP2也裂解半乳糖脂 和磷脂。此外，PTL明显需要胶脂酶，而PLRP2对胶脂酶的要求 仍然存在争议。我们的中心假设是，特定的结构域管理着 胰腺脂肪酶与底物的相互作用。要确定与确定 PTL和PLRP2的底物特异性，我们将利用有关结构和动力学性质的现有信息 这些脂肪酶。我们基于我们已发表的和初步的数据中的多个观察结果来采取这一方法。1) 结构研究和我们发表的PTL-Colipase复合体的研究指导了对PTL-Colipase复合体的鉴定 影响磷脂酶与PTL结合的不同结构域。2)结构数据分析和我们的初步研究 研究已经确定了影响这些物质吸收的PTL-Colipase和PLRP2中的关键残基 从脂肪酶到脂肪乳剂。3)PTL和PLRP2结构的比较和初步分析 确定了可以调节底物专一性差异的结构域。完成我们的具体目标 将提供胰腺脂肪酶与底物相互作用的每一步的分子细节。最终， 我们将能够合理地改造脂肪酶的酶性质，以满足特定的治疗或 工业需求。

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.