MECHANISM OF UREASE METALLOCENTER BIOSYNTHESIS

脲酶金属中心生物合成机制

• 批准号: 2405190
• 负责人: ROBERT P HAUSINGER
• 金额: $ 16.39万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2405190
• 关键词: Klebsiella; active sites; apoenzymes; bacterial proteins; carbon dioxide; enzyme biosynthesis; high performance liquid chromatography; metal metabolism; microorganism culture; microorganism metabolism; nickel; oligonucleotides; ultraviolet spectrometry; urease

DESCRIPTION: The long-term objective of this proposal is to characterize the biosynthesis of urease, a medically important enzyme that contains a novel binuclear-Ni active site. Formation of active enzyme requires the presence of the three urease subunits (UreA, UreB, and UreC), four accessory gene products (UreD, UreE, UreF, and UreG), carbon dioxide, and nickel ions. The four auxiliary proteins facilitate metal ion incorporation into the apoenzyme to form functional enzyme by as yet unclear processes. The CO2 is incorporated as a carbamate of a lysine that serves as a ligand to the metal center. Dr. Hausinger will elucidate the mechanism of metallocenter assembly in the best-characterized urease, that from Klebsiella aerogenes. His specific aims include: (1) purification and characterization of a complex that is comprised of UreD, UreF, and UreG, (2) formation of large quantities of a UreD-UreF-UreG-urease apoprotein complex, (3) examination of the activation properties of the UreD-UreF-UreG-urease apoprotein, (4) characterization of the UreE metallocenter and further investigation of whether this protein functions in Ni delivery to urease apoprotein, and (5) detailed analysis of the accessory protein-free activation process for urease apoprotein. The goal is to assess whether the accessory proteins function in any of the following roles: (1) providing specificity so that only Ni is bound to the apoprotein, (b) restricting the metal binding modes so that Ni can bind only in the proper coordination geometry, (c) catalyzing expulsion of non-Ni metal ions or incorrectly bound Ni ions, (d) aiding in the generation and/or delivery of CO2, and (e) assisting in the transfer of Ni from the presumed Ni carrier, UreE, to the apoprotein. This work on urease activation may serve as a model system for characterizing the mechanisms of metal incorporation into other metalloenzymes and it will greatly enhance our understanding of the biochemistry of Ni, an essential trace metal ion.

描述：该提议的长期目标是表征 尿素酶的生物合成，一种含有医学重要的酶，含有 新型的双核-NI活性位点。 活性酶的形成需要 三个尿素亚基（尿素，乌雷布和尿素）的存在，四个附件 基因产物（URE，URE，UREF和UREG），二氧化碳和镍离子。 四种辅助蛋白促进金属离子掺入到 载酶通过尚未清楚的过程形成功能酶。 二氧化碳是 掺入赖氨酸的氨基甲酸酯，可作为金属的配体 中心。 Hausinger博士将阐明金属中心的机制 从克雷伯氏菌的尿素酶中装配的尿素酶。 他的具体目的包括：（1）净化和表征 由UREF和UREG组成的复合物，（2）大型形成 URED-uref-ureg-rease蛋白酶蛋白复合物的数量，（3）检查 URED-uref-ureg-尿布蛋白的激活特性，（4） Uree金属中心的表征和进一步研究 该蛋白在Ni递送中是否起尿酶蛋白酶的作用，（5） 详细分析无辅助蛋白的激活过程 脲酶载脂蛋白。 目标是评估辅助蛋白是否 在以下任何角色中发挥作用：（1）提供特异性，以便 仅Ni与载脂蛋白结合，（b）限制金属结合模式 因此，Ni只能在适当的配位几何形状中结合，（c）催化 驱动非Ni金属离子或错误结合的ni离子，（d）帮助 二氧化碳的产生和/或交付，以及（e）协助转移 Ni从假定的Ni载体Uree到载脂蛋白。 这项工作 脲酶激活可以作为表征的模型系统 金属掺入其他金属酶的机理，它将 极大地增强了我们对NI生物化学的理解，这是必不可少的 痕量金属离子。