MECHANISM OF UREASE METALLOCENTER BIOSYNTHESIS

描述：本提案的长期目标是描述 尿素酶的生物合成，一种医学上重要的酶， 新的双核镍活性中心。 活性酶的形成需要 存在三种尿素酶亚基（UreA，UreB和UreC），四种辅助酶， 基因产物（UreD、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掺入到 脱辅基酶通过尚不清楚的过程形成功能酶。 将CO2 作为赖氨酸的氨基甲酸酯掺入，赖氨酸用作金属的配体 中心 豪辛格博士将阐明 组装在最好的特点，尿素酶，产气克雷伯氏菌。 他的具体目标包括：（1）纯化和表征 由UreD，UreF和UreG组成的复合物，（2）形成大的 一定量的UreD-UreF-UreG-尿素酶脱辅基蛋白复合物，（3） UreD-UreF-UreG-尿素酶脱辅基蛋白的活化特性，（4） 的特性和进一步的调查， 该蛋白质是否在Ni向尿素酶脱辅基蛋白的递送中起作用，以及（5） 详细分析了无辅助蛋白的活化过程， 尿素酶脱辅基蛋白 我们的目标是评估辅助蛋白是否 在以下任何角色中起作用：（1）提供特异性， 只有Ni与脱辅基蛋白结合，（B）限制金属结合模式 使得Ni只能以适当的配位几何结构结合，（c）催化 排出非Ni金属离子或不正确结合的Ni离子，（d）帮助 CO2的产生和/或输送，以及（e）协助 Ni从假定的Ni载体UreE到载脂蛋白。 这项工作对 尿素酶活化可以作为表征尿素酶活性的模型系统。 金属结合到其他金属酶的机制，它将 大大提高了我们对镍的生物化学的理解， 痕量金属离子。

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.