Contribution of salt bridge in the protease inhibitor SSI (Streptomyces subtilisin inhibitor) to its inhibitory action.
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-terminal 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.
Kojima
S
S
Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan.
Fujimura
K
K
Kumagai
I
I
Miura
K
K
D000409:Alanine; D000595:Amino Acid Sequence; D001120:Arginine; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008239:Lysine; D008715:Methionine; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013381:Subtilisins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D013816:Thermodynamics
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C018150:subtilisin inhibitor protein, Streptomyces; D001120:Arginine; D008715:Methionine; D013381:Subtilisins; D008239:Lysine; D000409:Alanine