Contribution of salt bridge in the protease inhibitor SSI (Streptomyces subtilisin inhibitor) to its inhibitory action

FEBS Letters

Kumagai, Izumi²  Fujimura, Konomi²  Miura, Kin-ichiro¹  Kojima, Shuichi¹ 

[1] Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171, Japan;Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Hongo, Tokyo 113, Japan

关键词: Protease inhibitor;    Protein engineering;    Electrostatic interaction;    Temporary inhibition;   

DOI  :  10.1016/0014-5793(94)80272-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The tertiary structure of proteinaceous protease inhibitors is considered to be maintained by various interactions in the molecule that prevent degradation by protease. In this study, the Arg²⁹ of Streptomyces subtilisin inhibitor (SSI) forming a salt bridge with the carboxyl group of carboxyl-tenninal Phe¹¹³ was replaced with Ala, Met or Lys by cassette mutagenesis to clarify the role of Arg²⁹ in the function of SSI. The inhibitory activity of each mutated SSI decreased with increasing incubation time after mixing with subtilisin, indicating that the SSI was changed into a temporary inhibitor upon mutation. This decrease was shown by SDS polyacrylamide gel electrophoresis to be due to cooperative degradation of the mutated SSI by subtilisin. In addition, the denaturation temperature of the Ala or Met mutant was decreased by ten degrees and that of the Lys mutant by 1.5 degrees, suggesting that the destabilization of SSI may be related to its temporary inhibition. Thus, interaction in the protease inhibitor molecule for maintaining the tertiary structure, such as that of Arg²⁹ in SSI, was shown to be required for the inhibitory action.

【 授权许可】

Unknown