Requirement for a disulfide bridge near the reactive site of protease inhibitor SSI (Streptomyces subtilisin inhibitor) for its inhibitory action.
The problem of why serine protease inhibitors having substrate-like structure around the reactive site are not degraded by the cognate protease has prompted us to investigate the structural requirements in Streptomyces subtilisin inhibitor (SSI) for its inhibitory action. We removed the disulfide bridge between Cys71 and Cys101 near the reactive site by oligonucleotide-directed mutagenesis, replacing both Cys residues with Ser residues. Inhibitory activity of the mutated SSI toward subtilisin BPN' was initially potent, but decreased remarkably with increasing incubation time after mixing (temporary inhibition), due to degradation of the mutated SSI by subtilisin via a specific intermediate with a nick at the reactive site (Met73-Val74). Binding affinity of subtilisin for the mutated SSI was reduced by more than 10(3)-fold, and the mutated SSI showed a 20 deg.C decrease in melting temperature, which probably mainly reflects the destruction of the region of alpha-helix containing Cys101. These results imply that the susceptibility of the mutated SSI to protease, and the irreversibility of the peptide bond cleavage at the reactive site, result from increased flexibility around the reactive site in the complex of the disulfide-bond-removed SSI with the protease, demonstrating the requirement of conformational rigidity around the reactive site of the inhibitor for its native inhibitory action.
Kojima
S
S
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Japan.
Kumagai
I
I
Miura
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine; D004220:Disulfides; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013381:Subtilisins / Q000037:antagonists & inhibitors*
D001426:Bacterial Proteins; D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C018150:subtilisin inhibitor protein, Streptomyces; D013381:Subtilisins; D003545:Cysteine