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Contribution of salt bridge in the protease inhibitor SSI ( Streptomyces subtilisin inhibitor) to its inhibitory action
Author(s) -
Kojima Shuichi,
Fujimura Konomi,
Kumagai Izumi,
Miura Kin-ichiro
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)80272-6
Subject(s) - subtilisin , serine protease , protease , protease inhibitor (pharmacology) , streptomyces , biochemistry , mutant , salt bridge , mutagenesis , enzyme inhibitor , site directed mutagenesis , chemistry , denaturation (fissile materials) , streptomycetaceae , proteases , inhibitory postsynaptic potential , enzyme , microbiology and biotechnology , biology , actinomycetales , bacteria , genetics , virus , neuroscience , nuclear chemistry , antiretroviral therapy , viral load , gene
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 Arg 29 of Streptomyces subtilisin inhibitor (SSI) forming a salt bridge with the carboxyl group of carboxyl‐tenninal Phe 113 was replaced with Ala, Met or Lys by cassette mutagenesis to clarify the role of Arg 29 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 Arg 29 in SSI, was shown to be required for the inhibitory action.