Catalytic Mechanisms and Biological Roles of Enzymes Involved in Energy Metabolism

结构决定功能。为了了解生命在单个分子水平上是如何工作的，生物化学家们识别生物分子的结构，以了解它们如何在活细胞中发挥作用。结构生物学领域基于三项实验技术：核磁共振光谱学、X射线结晶学和低温电子显微镜。每种方法都有局限性和优势。核磁共振波谱研究溶液中较小的大分子，在那里可以检测到它们的动力学。X射线结晶学要求分子排列成有序的晶体阵列。尽管存在这一限制，但对蛋白质数据库的搜索显示，几乎90%的模型来自X射线结晶学。在某种程度上，这是因为结晶学数据提供了更高分辨率的模型，即使是更大的大分子也能看到细节。低温电子显微镜对可以研究不同构象的大分子效果最好。最近探测器的改进使单分子的可视化具有与X射线结晶学相同的细节数量。我的研究计划专注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。我们研究具有三种或三种以上底物的复杂酶，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。酶是改善加拿大人健康的药物的主要靶点，了解它们是如何工作的指导新药的设计。这项建议的重点是确定两个相关的多亚单位酶，琥珀酰辅酶A合成酶和ATP柠檬酸裂解酶的结构。这两种酶都参与产生和使用能量，但它们还有额外的作用。琥珀酰辅酶A合成酶提供琥珀酰辅酶A，用于制造血红素。ATP-柠檬酸裂解酶提供乙酰辅酶A来调节基因的表达。我们理解酶是如何工作的方法是将酶与配体结晶。这些配体可以是反应的分子、反应的产物或阻止反应的分子。高分辨率模型使我们能够解释酶是如何催化反应的。当我们努力生长良好的晶体时，我们使用小角X射线散射和低温电子显微镜。我们的研究对于了解这些酶是如何工作的以及为什么琥珀酰辅酶A合成酶对于预防X连锁铁粒母细胞贫血是必要的是重要的。琥珀酰辅酶A合成酶不是药物靶点，但ATP-柠檬酸裂解酶是药物靶点。由于这两种酶是相关的，我们对每种酶如何发挥作用的洞察将有助于制药业设计抑制ATP-柠檬酸裂解酶的新药来治疗癌症或肥胖症。

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.