Imikhiqizo

Indlela imithelela yokuguqulwa kwezakhi zofuzo ehlangana ngayo ukuze iguqule umsebenzi we-enzymatic ayibikezeleki kalula ngoba le miphumela ayihlali ingeza ngendlela yomugqa.Ngakho-ke, ukubonakaliswa kwemithelela yokuguqulwa ngesikhathi esisodwa kwezinsalela ze-amino acid ezibopha i-substrate kungaba negalelo elikhulu ekuqondeni ukucaciswa kwe-substrate yama-enzyme.Ku-beta-glycosidase evela ku-Spodoptera frugiperda (Sfbetagly), zombili izinsalela ze-Q39 ne-E451 zisebenzisana ne-substrate futhi lokhu kubalulekile ekuchazeni ukucaciswa kwe-substrate.Izinguquko eziphindwe kabili ze-Sfbetagly (A451E39, S451E39 kanye ne-S451N39) zilungiswe i-mutagenesis eqondiswe kusayithi, evezwa kubhaktheriya futhi yahlanzwa kusetshenziswa i-affinity chromatography.Lawa ma-enzyme abonakala kusetshenziswa i-p-nitrophenyl beta-galactoside ne-p-nitrophenyl beta-fucoside njengama-substrates.Isilinganiso se-k cat/Km sokuguquguquka okukodwa nokuphindwe kabili kwe-Sfbetagly equkethe ukuguqulwa okuqondiswe kusayithi endaweni Q39 kanye ne-E451 yasetshenziswa ukubonisa ukuthi umphumela kumandla wamahhala we-ESdouble dagger (i-enzyme-transition state complex) yokuguqulwa okuphindwe kabili (Gdouble daggerxy) akusona isamba semiphumela ebangelwa ukuguqulwa okukodwa (i-Gdouble daggerx kanye ne-Gdouble daggery).Lo mehluko ku-Gdouble dagger ukhombisa ukuthi imiphumela yokuguqulwa okukodwa igqagqene kancane.Ngakho-ke, lo mphumela ojwayelekile ubala kanye kuphela ku-Gdouble daggerxy.Idatha ye-Crystallographic ku-beta-glycosidase yembula ukuba khona kwebhondi ye-hydrogen ye-bidentate efaka izinsalela ze-Q39 ne-E451 kanye neqembu elifanayo le-hydroxyl le-substrate.Ngakho-ke, kokubili idatha ye-thermodynamic ne-crystallographic iphakamisa ukuthi izinsalela ze-Q39 ne-E451 zinethonya elifanayo ekusebenzelaneni kwazo ne-substrate.