Thimet oligopeptidase: site‐directed mutagenesis disproves previous assumptions about the nature of the catalytic site

FEBS Letters

Stevens, Richard A.E.¹  Rawlings, Neil D.¹  Chen, Jinq-May¹  Wray, Paul W.¹  Barrett, Alan J.¹ 

[1] MRC Peptidase Laboratory, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK

关键词: Thimet oligopeptidase;    Metallopeptidase family M3;    Site-directed mutagenesis;    Zinc ligand;    DTT;    dithiothreitol;    IPTG;    isopropyl-β-d-thiogalactopyranoside;    QF02;    7-methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-Lys (2;    4-dinitrophenyl);   

DOI  :  10.1016/S0014-5793(98)01032-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Zinc metallopeptidases that contain the His-Glu-Xaa-Xaa-His (HEXXH) motif generally have a third ligand of the metal ion that may be either a Glu residue (in clan MA) or a His residue (in clan MB) (Rawlings and Barrett (1995) Methods Enzymol. 248, 183–228). Thimet oligopeptidase has not yet been assigned to either clan, and both Glu and His residues have been proposed as the third ligand. We mutated candidate ligand residues in the recombinant enzyme and identified Glu, His and Asp residues that are important for catalytic activity and/or stability of the protein. However, neither of the Glu and His residues close to the HEXXH motif that have previously been suggested to be ligands is required for the binding of zinc. We conclude that thimet oligopeptidase is not a member of clan MA or clan MB and it is likely that the enzyme possesses a catalytic site and protein fold different from those identified in any metallopeptidase to date. The definitive identification of the third zinc ligand may well require the determination of the crystallographic structure of thimet oligopeptidase or one of its homologues.

【 授权许可】

Unknown