Alteration of inhibitory properties of Pleurotus ostreatus proteinase A inhibitor 1 by mutation of its C-terminal region.
Pleurotus ostreatus proteinase A inhibitor 1 (POIA1), which is composed of 76 residues without disulfide bridges, is a unique inhibitor in that it exhibits sequence similarity to the propeptides of subtilisins. In order to elucidate the inhibitory mechanism of POIA1, we constructed an expression system for a synthetic POIA1 gene. The wild-type POIA1 was found to inhibit subtilisin BPN' with an inhibitor constant (K(i)) of 3.2 x 10(-9) M, but exhibited a time-dependent decrease of inhibitory activity as a consequence of degradation by the protease, showing that the wild-type POIA1 was a temporary inhibitor when subtilisin BPN' was used as a target protease. Since POIA1 shows sequence similarity to the propeptide of subtilisin, which is known to inhibit the protease via its C-terminal region, the C-terminal six residues of POIA1 were replaced with those of the propeptide of subtilisin BPN'. The mutated POIA1 inhibited subtilisin BPN' with a K(i) value of 2.8 x 10(-11) M and did not exhibit time-dependent decrease of inhibitory activity, showing about 100-fold increases in binding affinity for, and resistance to, the protease. These results clearly indicate that the C-terminal region of POIA1 plays an important role in determining the inhibitory activity toward the protease, and that the increase in binding ability to the protease is closely related to resistance to proteolytic degradation. Therefore, the inhibitory properties of POIA1 can be altered by mutation of its C-terminal region.
Kojima
S
S
Institute for Biomolecular Science, Gakushuin University, Tokyo, Mejiro, 171-8588, Japan. shuichi.kojima@gakushuin.ac.jp
Hisano
Y
Y
Miura
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology*; D011480:Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000235:genetics; D014162:Transfection
D004792:Enzyme Precursors; D005656:Fungal Proteins; C018134:PAI3 protein, S cerevisiae; D010446:Peptide Fragments; D011480:Protease Inhibitors; D029701:Saccharomyces cerevisiae Proteins; C057107:prosubtilisin; D013381:Subtilisins