1K7I

PrtC from Erwinia chrysanthemi: Y228F mutant

1K7I の概要

• エントリーDOI: 10.2210/pdb1k7i/pdb
• 分子名称: secreted protease C, CALCIUM ION, ZINC ION, ... (4 entities in total)
• 機能のキーワード: metalloprotease, hydrolase, protease
• 由来する生物種: Erwinia chrysanthemi
• 細胞内の位置: Secreted: P16317
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 51965.20

構造登録者

Baumann, U.,Hege, T. (登録日: 2001-10-19, 公開日: 2002-10-19, 最終更新日: 2024-02-07)

主引用文献

Hege, T.,Baumann, U.
Protease C of Erwinia chrysanthemi: The Crystal Structure and Role of Amino Acids Y228 and E189
J.Mol.Biol., 314:187-193, 2001

PubMed Abstract: PrtC, a metallo-protease secreted by Erwinia chrysanthemi, is a member of the serralysin family and hence belongs to the metzincin superfamily. While the crystal structures of representatives of all metzincin subfamilies have been elucidated in the past, there is still some controversy about the reaction mechanism and the role of certain characteristic amino acids in the active centre. In this study, we probed the role of Tyr228 and Glu189 by site-directed mutagenesis and X-ray crystallography. There is evidence that these residues participate in catalysis, although conflicting hypotheses have been proposed. The crystal structures of wild-type and mutants have been refined to an R(free) of about 0.20 at resolutions of 2.0 A or better. Exchange of Glu189 versus alanine reduces the catalytic efficiency to less than 0.5 % using resorufin casein as substrate and to about 3 % using an assay utilising the thiol ester Ac-Pro-Leu-Gly-[(S)Leu]-Leu-Gly-OEt. The drop in activity is caused by a reduction in k(cat) while the K(M) values are virtually the same. In the resorufin casein assay, the mutant Y228F shows about 3 % of the wild-type activity and in the thiol ester assay this increases to about 56 %. In the latter case, the K(M) value of the mutant is increased from 5.3 mM to 9.0 mM with only little reduction in k(cat). The different behaviour of this mutant with respect to the two substrates can be explained by a switch in the rate-determining step during catalysis. The study presented here provides clear evidence that Glu189 of the HEXXHXXGXXH motif is the catalytic base, while Tyr228 is more likely involved in substrate binding and the stabilisation of the tetrahedral transition state.

PubMed: 11718553
DOI: 10.1006/jmbi.2001.5124

実験手法

X-RAY DIFFRACTION (1.59 Å)