The Mechanism Study On Sedoheptulose-1,7-bisphosphatase(SBPase) From Arabidopsis Catalyze Substrates SBP And FBP

Sedoheptulose-1,7-bisphosphatase(SBPase)main exsitance in chloroplast,is a vital regulation enzyme in photosynthesis of higher plant,in the photosynthesis dark reaction,it can catalyze substrate Sedoheptulose-1,7-bisphosphate(SBP)to inorganic phosphate and Sedoheptulose-7-phosphate.Arabidopsis thaliana due to its small genome,shor-time life circle,become to model plant of scientific research..It is benefit for us to develop the new herbicide based on SBPase through knowing the Arabidopsis thaliana SBPase structure,its functions and catalyze mechanism.The study mainly covers four aspects:(1)Construct Ar-SBPase vector and the enzyme's expression and purification:we make full use of genetic methods to construct a recombination of pCold II-Ar-SBPase.It proved by the experiments that used pCold ? as expression vector and PG-TF2 as expression bacteria,we can get 2.4mg/L,catalyze active5.34U/mg,high solubility and high active Ar-SBPase.(2)Arabidopsis thaliana Sedoheptulose-1,7-bisphosphatase(Ar-SBPase)can not only catalyze the substrate SBP but also can catalyze the substrate FBP,by the test,we found kinetic parameter of Ar-SBPase catalyze the substrate SBP was that 37?,pH8.8,reaction time was 6min,DTT concentrate was 1mM,Ka with Mg2+ was 2.238 mM,Km with SBP was 0.2607 mM.Meanwhile Ar-SBPase catalyze the substrate FBP 37?,pH8.8,reaction time was 8min,DTT concentrate was 15mM,Ka with Mg2+was 2.415 mM,Km with FBP was 0.2217 mM.Compared the datas we could found Ar-SBPase catalyze the two substrate has quite different catalyze active,besides that,the other big difference is the sensitivity to the deoxidizer concentrate.(3)We make the comparison of Ar-SBPase,the Ri-SBPase and Cy FBP/SBPase,,after that we used the human FBP active cavity as molecular cavity,TOX protein as template to make the homology modeling,finalyy we found thirteen key amino acids R267,N265,N312,Y291,K321,R323,E327,D180,D177,D127,S182,R290,D298 and mutants all of them to alanine.It showed compared with wild-type,R267A,N265A,N312A,Y291A,E327A,D180A,D177A,D127A these eight amino acids lost the catalyze active,N312A,K321A,R323A catalyze active declined almost 90%,D298A active declined 20%,only S182A active increased 10%.It also can prove the modeling and experiment results.(4)Take the ArabidopsisFBP/SBPase as a target to screen the compounds.It is found by experiment that compounds D13-tqd-150,D13-tqd-151,D13-tqd-152 had a high inhibit rate to Ar-SBPase catalyze substrate SBP.Their IC50 seperately is 7.27?M,7.05?M and 8.99?M.This three moleculars has the same cage construction,therefore,we can optimize the construction to screen the more efficient inhibitors.