A structure‐based site‐directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis

FEBS Letters

de Camargo, Antonio C.M³  Araújo, Maurı́cio C¹  Juliano, Luiz⁴  Oliveira, Vitor⁴  Tersariol, Ivarne L.S²  Ferro, Emer S¹  Rioli, Vanessa¹  Juliano, Maria A⁴ 

[1] Departamento de Histologia e Embriologia, Programa de Biologia Celular, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo, SP, Brazil;Centro Interdisciplinar de Investigação Bioquı́mica (CIIB), Universidade de Mogi das Cruzes, 08780-911 Mogi das Cruzes, SP, Brazil;Centro de Toxinologia Aplicada (CAT), Instituto Butantan, 05467-010 São Paulo, SP, Brazil;Departamento de Biofı́sica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil

关键词: Enzyme specificity;    Catalytic mechanism;    Site-directed mutagenesis;    Abz;    ortho-aminobenzoic acid;    EDDnp;    N-(2;    4-dinitrophenyl) ethylenediamine;   

DOI  :  10.1016/S0014-5793(03)00310-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr⁶¹³ (EP24.16) or Tyr⁶¹² (EP24.15) to either Phe or Ala promoted a strong reduction of k _cat/K _M for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k _cat/K _M of 2×10⁵ M⁻¹ s⁻¹ for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k _cat/K _M=3×10⁵ M⁻¹ s⁻¹) which contains Gly at the corresponding position; the wild type EP24.15 has a k _cat/K _M of 2.5×10⁴ M⁻¹ s⁻¹ for this substrate.

【 授权许可】

Unknown