定点诱变G6PD基因的表达及突变酶的生物学功能研究

首次将9种人工定点诱变的G6PD基因转化至G6PD缺陷的大肠杆菌HB351(DE3)中表达,并对突变酶的生物学功能进行研究。初步证实G6PD基因m1376 G to T(Arg 459keu),1388 G to A(Arg 463 His)突变可降低酶活性并引起酶动力学改变。这可能与取代氨基酸的化学结构、所带电荷的性质及极性有关。这两个部位的精氨酸在酶与NADP~ 的结合过程中亦起到重要作用。赖氨酸取代精氨酸对酶与NADP~ 的结合影响不大。引入无义突变,证实G6PD第459位以后的氨基酸对酶活性有重要影响。

Expression and Biochemical Characterization of Human G6PD Gene 1376 and 1388 Mutation in G6PD-deficient Escherichia coli

Nine types of human G6PD gene mutated at the positions of nt 1376 and nt1388 by site-directed mutagenesis were transformed into the strain of G6PD dificent E.coli HB 351(DE3). The mutated gene was expressed successfully and the enzymekinetic studies undertaken according to WHO standarzation. The results showed thatthe arginine residues at the positions of 459 and 463 of G6PD gene play animportant role in maintaining activity of the enzyme. The amino acid structure, polarity,and electronic property may be responsible for it. The arginine residues at thepositions of 459 and 463 are also important for the enzyme-NADP~ binding, but itwas not interfered by the lysine-arginine substitution. By inducing a non-sensemutation, it was further demonstrated that the amino acids residueds behind theposition of 459 were extremelv significant for G6PD activity