DSpace university logo mark
詳細検索
Japanese | English 

NAOSITE : Nagasaki University's Academic Output SITE > 050 薬学部 > 050 学術雑誌論文 >

Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase.


ファイル 記述 サイズフォーマット
JMB396_1081.pdf1.31 MBAdobe PDF本文ファイル

タイトル: Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase.
著者: Yamashita, Kinuyo / Nakajima, Yoshitaka / Matsushita, Hayato / Nishiya, Yoshiaki / Yamazawa, Ryuji / Wu, Yu-Fan / Matsubara, Futoshi / Oyama, Hiroshi / Ito, Kiyoshi / Yoshimoto, Tadashi
発行日: 2010年 3月 5日
出版者: Elsevier Ltd.
引用: Journal of molecular biology, 396(4), pp.1081-1096; 2010
抄録: Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity. We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl(2) or MnCl(2). In the E122Q mutant, k(cat) is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.
キーワード: amidohydrolase superfamily / binuclear metal center / catalytic mechanism / creatininase / crystal structure
URI: http://hdl.handle.net/10069/22949
ISSN: 00222836
DOI: 10.1016/j.jmb.2009.12.045
PubMed ID: 20043918
権利: Copyright © 2009 Elsevier Ltd All rights reserved.
資料タイプ: Journal Article
原稿種類: author
出現コレクション:050 学術雑誌論文

引用URI : http://hdl.handle.net/10069/22949

このリポジトリに保管されている文献はすべて著作権により保護されています。
印刷やダウンロード等データの複製は、調査研究・教育または学習を目的とする場合に限定されます。

 

Valid XHTML 1.0! Copyright © 2006-2015 長崎大学附属図書館 - お問い合わせ Powerd by DSpace