Catalytic Mechanisms and Biological Roles of Enzymes Involved in Energy Metabolism

结构决定功能。为了在单个分子的层面上理解生命是如何运作的，生化学家要辨别生物分子的结构，以了解它们在活细胞中是如何起作用的。结构生物学领域基于三种实验技术：核磁共振波谱学、x射线晶体学和低温电子显微镜。每种方法都有其局限性和优点。核磁共振波谱学观察溶液中较小的大分子，在那里它们的动力学可以被检测到。x射线晶体学要求分子排列成有序的晶体阵列。尽管存在这种限制，但对蛋白质数据库的搜索表明，几乎90%的模型来自x射线晶体学。在某种程度上，这是因为晶体学数据提供了更高分辨率的模型，甚至可以看到更大的大分子的细节。低温电子显微镜最适合研究不同构象的大分子。探测器的最新改进使单个分子的可视化具有与x射线晶体学相同数量的细节。******我的研究项目侧重于使用结构生物学的工具来研究酶，以发现酶如何发挥其生物学作用。我们研究具有三个或更多底物的复杂酶，以了解这些具有多个活性位点的多亚基酶如何在活细胞中催化它们的反应。酶是用于改善加拿大人健康的药物的主要目标，了解它们如何起作用可以指导新药的设计。******本提案的重点是确定两个相关的多亚基酶，琥珀酰辅酶a合成酶和atp -柠檬酸水解酶的结构。这两种酶都参与能量的产生和利用，但它们还有其他作用。琥珀酰辅酶a合成酶提供用于制造血红素的琥珀酰辅酶a。atp -柠檬酸裂解酶提供乙酰辅酶a来调节基因的表达。我们理解酶如何工作的方法是用配体使酶结晶。这些配体可以是反应的分子，反应的产物或者是阻止反应的分子。高分辨率模型使我们能够解释酶是如何催化反应的。当我们努力生长好的晶体时，我们使用小角度x射线散射和低温电子显微镜。******我们的研究对于理解这些酶是如何工作的以及为什么琥珀酰辅酶a合成酶是预防x连锁铁母细胞性贫血所必需的非常重要。琥珀酰辅酶a合成酶不是药物靶标，但atp -柠檬酸解酶是。由于这两种酶是相关的，我们对每一种酶如何工作的洞察将有助于制药行业设计出抑制atp -柠檬酸裂解酶的新药，以治疗癌症或肥胖。

Structure dictates function. To understand how life works at the level of individual molecules, biochemists discern structures of biological molecules to learn how they function in a living cell. The field of structural biology is based on three experimental techniques: nuclear magnetic resonance spectroscopy, X-ray crystallography and cryoelectron microscopy. Each has limitations and advantages. Nuclear magnetic resonance spectroscopy looks at smaller macromolecules in solution where their dynamics can be detected. X-ray crystallography requires that the molecules be arranged in the ordered array of a crystal. Despite this limitation, a search of the Protein Data Bank shows that almost 90% of models are from X-ray crystallography. In part, this is because crystallographic data provide models of higher resolution, allowing details to be seen for even the larger macromolecules. Cryoelectron microscopy works best with large macromolecules whose different conformations can be investigated. Recent improvements in detectors allow single molecules to be visualized with the same amount of detail as from X-ray crystallography.******My research program focuses on investigating enzymes using the tools of structural biology to discover how the enzymes perform their biological roles. We study complex enzymes that have three or more substrates to understand how these multi-subunit enzymes with multiple active sites catalyze their reactions in living cells. Enzymes are a major target of drugs taken to improve the health of Canadians and understanding how they work guides the design of new drugs.******This proposal focuses on determining structures of two related multi-subunit enzymes, succinyl-CoA synthetase and ATP-citrate lyase. Both enzymes are involved in producing and using energy, but they have additional roles. Succinyl-CoA synthetase provides succinyl-CoA, which is used to make heme. ATP-citrate lyase provides acetyl-CoA to regulate the expression of genes. Our approach to understanding how enzymes work is to crystallize the enzyme with ligands. These ligands could be the molecules that react, products of the reaction or molecules that prevent the reaction. High-resolution models allow us to interpret how the enzyme catalyzes the reaction. When we struggle to grow good crystals, we use small angle X-ray scattering and cryoelectron microscopy.******Our research is important for understanding how these enzymes work and why succinyl-CoA synthetase is necessary to prevent X-linked sideroblastic anemia. Succinyl-CoA synthetase is not a drug target but ATP-citrate lyase is. Since the two enzymes are related, our insight into how each works will help the pharmaceutical industry design new drugs that inhibit ATP-citrate lyase to treat cancer or obesity.