Nā huahana

ʻAʻole hiki ke ʻike maʻalahi ʻia ke ʻano o ka hoʻololi ʻana i ka hana enzymatic no ka mea ʻaʻole hoʻohui mau kēia mau hopena i ke ʻano laina.No laila, ʻo ke ʻano o nā hopena o ka hoʻololi like ʻana o nā koena amino acid e hoʻopaʻa ai i ka substrate hiki ke hana i kahi kōkua nui i ka hoʻomaopopo ʻana i ke ʻano kikoʻī o nā enzyme.I loko o ka beta-glycosidase mai Spodoptera frugiperda (Sfbetagly), pili nā koena ʻelua Q39 a me E451 me ka substrate a he mea nui kēia no ka wehewehe ʻana i ka kikoʻī o ka substrate.Ua hoʻomākaukau ʻia nā mutagenes ʻelua o Sfbetagly (A451E39, S451E39 a me S451N39) e ka mutagenesis kuhikuhi i ka pae, i hōʻike ʻia i loko o ka bacteria a hoʻomaʻemaʻe ʻia me ka hoʻohana ʻana i ka chromatography affinity.Hoʻohālikelike ʻia kēia mau enzyme me ka hoʻohana ʻana i ka p-nitrophenyl beta-galactoside a me ka p-nitrophenyl beta-fucoside ma ke ʻano he substrates.Ua hoʻohana ʻia ka lakio k cat/Km no nā mutants hoʻokahi a me ʻelua o Sfbetagly i loaʻa nā hoʻololi i kuhikuhi ʻia i ka pūnaewele ma nā kūlana Q39 a me E451 e hōʻike i ka hopena i ka ikehu manuahi o ESdouble dagger (enzyme-transition state complex) o nā hoʻololi pālua (Gdouble daggerxy) ʻaʻole ia ka huina o nā hopena i loaʻa mai nā hoʻololi hoʻokahi (Gdouble daggerx a me Gdouble daggery).ʻO kēia ʻokoʻa i loko o Gdouble dagger e hōʻike ana i nā hopena o ka hoʻololi ʻana hoʻokahi i uhi ʻia.No laila, helu ʻia kēia hopena maʻamau i hoʻokahi wale nō ma Gdouble daggerxy.Hōʻike ka ʻikepili crystallographic ma nā beta-glycosidases i ka loaʻa ʻana o kahi paʻa hydrogen bidentate e pili ana i nā koena Q39 a me E451 a me ka hui hydroxyl like o ka substrate.No laila, hōʻike ka ʻikepili thermodynamic a me ka crystallographic e hoʻoikaika like nā koena Q39 a me E451 i kā lākou pilina me ka substrate.