Impact of the lysine‐188 and aspartic acid‐189 inversion on activity of trypsin

FEBS Letters

Haertlé, Thomas²  Declerck, Nathalie⁴  Mollé, Daniel³  Chobert, Jean-Marc²  Gantier, René²  Briand, Loı̈c²  Tran, Vinh¹  Léonil, Joëlle³ 

[1] LPCM, Nantes, France;Institut National de la Recherche Agronomique, LEIMA, P.O. Box 71627, 44316 Nantes Cedex 3, France;LRTL, Rennes, France;INA-PG, Thiverval-Grignon, France

关键词: Trypsin;    Site-directed mutagenesis;    Synthetic substrate;    Protease;   

DOI  :  10.1016/S0014-5793(98)01611-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The impact of the charge rearrangement on the specificity of trypsin was tested by an inversion of sequence K188D/D189K maintaining the integrity of the charges of the substrate binding pocket when switching their polarity. In native trypsin, aspartate 189 situated at the bottom of the primary substrate binding pocket interacts with arginine and lysine side chains of the substrate. The kinetic parameters of the wild-type trypsin and K188D/D189K mutant were determined with synthetic tetrapeptide substrates. Compared with trypsin, the mutant K188D/D189K exhibits a 1.5- to 6-fold increase in the K _m for the substrates containing arginine and lysine, respectively. This mutant shows a ∼30-fold decrease of its k _cat and its second-order rate constant k _cat/K _m decreases ∼40- and 150-fold for substrates containing arginine and lysine, respectively. Hence, trypsin K188D/D189K displays a large increase in preference for arginine over lysine.

【 授权许可】

Unknown