期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：20
页码：11140-11144
DOI：10.1073/pnas.96.20.11140
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Syntheses of metal-containing enzymes often require the participation of accessory proteins. The roles played by many of these accessory proteins are poorly characterized. Klebsiella aerogenes urease, a nickel-containing enzyme, provides an ideal system to study metallocenter assembly. Here, we describe a method for isolating a complex containing urease apoprotein and the UreD, UreF, and UreG accessory proteins. We demonstrate that urease apoprotein in this complex is activated to near wild-type enzyme levels when incubated with nickel ions and high ({approx}100 mM) concentrations of bicarbonate. Significantly, we also observed nickel-dependent activation at physiologically relevant ({approx}100 {micro}M) bicarbonate levels, but only in the presence of GTP. Based on studies involving a nonhydrolyzable analog of GTP, we conclude that nucleotide hydrolysis, not just binding, is required for this process. The critical nucleotide-binding site was localized to UreG on the basis of experiments using a variant complex. These studies highlight the relevance of the UreD-UreF-UreG-urease apoprotein complex to nickel metallocenter assembly and explain the previously identified in vivo energy requirement for urease activation.
