Active Site Mechanisms of Free Radical Metalloenzymes

描述(由申请人提供)：自由基氧化铜酶作为一个广泛的家族正在出现，跨越了深层次的生物分裂。这些酶包括真菌酶半乳糖氧化酶和乙二醛氧化酶，其主要特征是存在一个由铜离子和内在氧化还原辅因子组成的金属络合物。蛋白质来源的辅因子酪氨酰半胱氨酸(Tyr-Cys)是由共价交联氨基酸侧链形成的，经过可逆氧化生成稳定的蛋白质自由基，在催化过程中起着至关重要的作用。 本研究旨在结合化学猝灭(CQ)、快速冷冻猝灭(RFQ)、同位素动力学和自旋捕获实验来阐明半乳糖氧化酶中Tyr-Cys辅因子的生物生成机制。质谱学将用于确定共价蛋白质修饰的位置和结构。巴斯德毕赤酵母营养缺陷型菌株将被用来表达含有非天然氨基酸的半乳糖氧化酶，作为辅因子生物发生和金属代谢机制的探针。对蛋白质中Tyr-Cys辅助因子的氚标记将允许使用EPR光谱分析Tyr-Cys自由基中未配对的电子分布。对Tyr-Cys自由基位点的计算实验将提供对这一独特催化特性的深入了解。来自广泛不同王国的选定模式生物的自由基铜氧化酶将被表达和表征，以确定这种不寻常的结构在生物学中的范围和作用。 这项工作将解决金属结构和机制的重要元素，并在更大的背景下，将有助于理解金属介导的蛋白质修饰的机制，这是一个生化过程，是包括细胞色素C氧化酶在内的重要酶成熟的必要步骤。

DESCRIPTION (provided by applicant): Radical copper oxidases are emerging as a broad family of enzymes spanning deep biological divisions. The defining feature of these enzymes, which include the fungal enzymes galactose oxidase and glyoxal oxidase, is the presence of a metalloradical complex comprised of a copper ion and an intrinsic redox cofactor. The protein-derived cofactor, tyrosyl-cysteine (Tyr-Cys) is formed by covalent cross-linking amino acid side chains and undergoes reversible oxidation to a stable protein free radical that plays an essential role in catalysis. This proposal aims to elucidate the mechanism of Tyr-Cys cofactor biogenesis in galactose oxidase, using a combination of Chemical Quench (CQ), Rapid Freeze Quench (RFQ), isotope kinetics and spin trapping experiments. Mass spectrometry will be used to define the sites and structures of covalent protein modifications. Auxotrophic strains of Pichia pastoris will be used to express galactose oxidase incorporating non-natural amino acids as probes of cofactor biogenesis and metalloradical mechanisms. Deuterium labeling of the Tyr-Cys cofactor in the protein will permit the unpaired electron distribution in the Tyr-Cys free radical to be mapped using EPR spectroscopy. Computational experiments on the Tyr-Cys free radical site will provide insight into the properties of this unique catalytic feature. Radical copper oxidases from selected model organisms in widely divergent kingdoms will be expressed and characterized to define the range and roles of this unusual structure in biology. This work will resolve important elements of metalloradical structure and mechanism, and, in a larger context, will contribute to understanding mechanisms of metal-mediated protein modification, a biochemical process that is a necessary step in the maturation of important enzymes including cytochrome c oxidase.