1A5M

K217A VARIANT OF KLEBSIELLA AEROGENES UREASE

1A5M の概要

• エントリーDOI: 10.2210/pdb1a5m/pdb
• 分子名称: UREASE (GAMMA SUBUNIT), UREASE (BETA SUBUNIT), UREASE (ALPHA SUBUNIT), ... (4 entities in total)
• 機能のキーワード: hydrolase (urea amido), mutant, nickel metalloenzyme, hydrolase
• 由来する生物種: Klebsiella aerogenes; 詳細
• 細胞内の位置: Cytoplasm (By similarity): P18316 P18315 P18314
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 82403.68

構造登録者

Pearson, M.A.,Schaller, R.A.,Michel, L.O.,Karplus, P.A.,Hausinger, R.P. (登録日: 1998-02-17, 公開日: 1998-05-27, 最終更新日: 2024-02-07)

主引用文献

Pearson, M.A.,Schaller, R.A.,Michel, L.O.,Karplus, P.A.,Hausinger, R.P.
Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand.
Biochemistry, 37:6214-6220, 1998

PubMed Abstract: Klebsiella aerogenes urease possesses a dinuclear metallocenter in which two nickel atoms are bridged by carbamylated Lys217. To assess whether carbamate-specific chemistry is required for urease activity, site-directed mutagenesis and chemical rescue strategies were combined in efforts to place a carboxylate group at the location of this metal ligand. Urease variants with Lys217 replaced by Glu, Cys, and Ala (K217E, K217C/C319A, and K217A proteins) were purified, shown to be activated by incubation with small organic acids plus Ni(II), and structurally characterized. K217C/C319A urease possessed a second change in which Cys319 was replaced by Ala in order to facilitate efforts to chemically modify Cys217; however, this covalent modification approach did not produce active urease. Chemical rescue of the K217E, K217C/C319A, and K217A variants required 2, 2, and 10 h, respectively, to reach maximal activity levels. The highest activity generated [224 micromol of urea degraded.min-1.(mg of protein)-1, for K217C/C319A urease incubated with 500 mM formic acid and 10 mM Ni at pH 6.5] corresponded to 56% of that measured for in vitro activation of the wild-type apoprotein. While the K217E apoprotein showed minimal structural perturbations, the K217C/C319A apoprotein showed a disordering of some active site residues, and the K217A apoprotein revealed a repositioning of His219 to allow the formation of a hydrogen bond with Thr169, thus replacing the hydrogen bond between the amino group of Lys217 and Thr169 in the native enzyme. Importantly, these structures allow rationalization of the relative rates and yields of chemical rescue experiments. The crystal structures of chemically rescued K217A and K217C/C319A ureases revealed a return of the active site residues to their wild-type positions. In both cases, noncovalently bound formate was structurally equivalent to the Lys-carbamate as the bridging metallocenter ligand. We conclude that carbamate-specific chemistry is not required for urease catalysis.

PubMed: 9558361
DOI: 10.1021/bi980021u

実験手法

X-RAY DIFFRACTION (2 Å)