SELENOSUBTILISINS PEROXIDASE-ACTIVITY DOES NOT REQUIRE AN INTACT OXYANION HOLE

Replacement of the active site serine in subtilisin with selenocystein e converts the protease to a peroxidase. The contribution of the oxyan ion hole to the efficiency of this novel activity has been investigate d by substituting Asn155 with glycine via site-directed mutagenesis. A lthough the side chain amide of Asn155 provides a hydrogen bond that i ncreases the efficiency of the hydrolytic reactions promoted by native subtilisin, it appears to contribute relatively little to the stabili ty of key intermediates or transition states of the selenosubtilisin-c atalyzed peroxidase reaction. Instead, the modest changes in kinetic p roperties resulting from mutation of the selenoenzyme can be explained by subtle alterations in substrate binding at the S1 pocket which per turb the complex equilibria that determine forward progress along the reaction coordinate. Additional modifications of the subtilisin bindin g site will apparently be necessary to fully exploit the catalytic pot ential of the oxyanion hole for selenium-dependent redox chemistry. (C ) 1997 Elsevier Science Ltd.