Iimveliso

Indlela ekudityaniswa ngayo iziphumo zenguqu ngaxeshanye ukuguqula umsebenzi we-enzymatic ayiqikeleleki lula kuba ezi ziphumo azisoloko zisongeza ngendlela elandelelanayo.Ngenxa yoko, ukubonakaliswa kweempembelelo zokuguqulwa kwangaxeshanye kweentsalela ze-amino acid ezibopha i-substrate ingenza igalelo elibalulekileyo ekuqondeni kwe-substrate ethile ye-enzymes.Kwi-beta-glycosidase esuka kwi-Spodoptera frugiperda (Sfbetagly), zombini iintsalela ze-Q39 kunye ne-E451 zisebenzisana ne-substrate kwaye oku kubalulekile ekuchazeni i-substrate ethile.Iinguqu eziphindwe kabini ze-Sfbetagly (A451E39, S451E39 kunye ne-S451N39) zilungiswe yi-site-directed mutagenesis, echazwe kwiibhaktheriya kwaye yacocwa ngokusebenzisa i-affinity chromatography.Ezi enzymes zibonakaliswe ngokusebenzisa i-p-nitrophenyl beta-galactoside kunye ne-p-nitrophenyl beta-fucoside njengama-substrates.I-k cat/Km ratio ye-single kunye ne-double mutants ye-Sfbetagly equlathe ukuguqulwa kwesiza-directed kwizikhundla ze-Q39 kunye ne-E451 yasetyenziswa ukubonisa ukuba isiphumo kumandla wasimahla we-ESdouble dagger (i-enzyme-transition state complex) yokuguqulwa kabini (Gdouble daggerxy) ayisosixa seziphumo ezibangelwa kutshintsho olunye (i-Gdouble daggerx kunye ne-Gdouble daggery).Lo mahluko kwi-Gdouble dagger ubonisa ukuba iimpembelelo zotshintsho lwenguqu enye zihamba ngokuyinxenye.Ke, esi siphumo siqhelekileyo sibala kanye kuphela kwi-Gdouble daggerxy.Idatha ye-Crystallographic kwi-beta-glycosidases ityhila ubukho be-bidentate hydrogen bond ebandakanya iintsalela ze-Q39 kunye ne-E451 kunye neqela elifanayo le-hydroxyl ye-substrate.Ke ngoko, zombini idatha ye-thermodynamic kunye necrystallographic icebisa ukuba iintsalela ze-Q39 kunye ne-E451 zenza impembelelo efanayo ekusebenzisaneni kwazo kunye ne-substrate.