MECHANISM OF UREASE METALLOCENTER BIOSYNTHESIS

描述：这项提案的长期目标是 尿素酶的生物合成，这是一种医学上重要的酶，含有 新的双核镍活性中心。活性酶的形成需要 存在三个尿素酶亚基(尿素、尿素B和UREC)，四个附件 基因产物(UreE、UreF和UreG)、二氧化碳和镍离子。 这四种辅助蛋白促进金属离子进入细胞内 辅酶通过尚不清楚的过程形成功能酶。二氧化碳是 以赖氨酸的氨基甲酸酯形式存在，用作金属的配体 中间。豪辛格博士将阐明金属分配中心的机制 装配在最具特性的尿素酶中，即产气克雷伯菌的尿素酶。 他的具体目标包括：(1)纯化和表征一种 由ORED、UREF和UREG组成的复合体，(2)形成大型 UreF-UreG-Urease脱辅基蛋白复合体的数量，(3)检测 UreD-UreF-UreG-Urease脱辅基蛋白的激活特性，(4) UreE金属配位中心的表征及进一步研究 该蛋白是否在镍转运到尿素酶脱辅基蛋白中起作用，以及(5) 详细分析了辅助性无蛋白激活过程 尿素酶脱辅基蛋白。我们的目标是评估辅助蛋白是否 在以下任一角色中发挥作用：(1)提供特殊性，以便 只有镍与脱脂蛋白结合，(B)限制金属结合模式 这样镍只能以合适的配位几何构型键合，(C)催化 排出非镍金属离子或不正确结合的镍离子，(D)协助 二氧化碳的产生和/或输送，以及(E)协助转移 镍从假定的镍载体UreE到脱辅基蛋白。这项工作是关于 尿素酶的激活可以作为一种模型系统来表征 金属与其他金属酶结合的机制和它将 大大提高了我们对镍的生物化学的了解，镍是一种重要的 微量金属离子。

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.