pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15)

FEBS Letters

Wolfson, Adele J¹  Glucksman, Marc J²  Edwards, Sarah R¹  Pabon, Amanda²  Sigman, Jeffrey A¹ 

[1] Department of Chemistry, Wellesley College, Wellesley, MA 02481, USA;FUHS/Chicago Medical School, Midwest Proteome Center, Chicago, IL, USA

关键词: Homology modeling;    Thimet oligopeptidase;    Metal activation;    pH dependence;    Transition state stabilization;    Tyrosine;    TOP;    thimet oligopeptidase;    MCA;    7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-Lys-dinitrophenol;    DTT;    dithiothreitol;    cFP;    N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate;    CPA;    carboxypeptidase A;   

DOI  :  10.1016/S0014-5793(03)00548-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clarifies the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pK _a of 5.7 and a basic limb with pK _a of 8.8. The acidic limb can be attributed to protonation of a residue affecting k _cat while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin.

【 授权许可】

Unknown