青霉素G酰化酶β亚基Ser^(579)、Arg^(580)的定点突变研究  

STUDIES ON THE FUNCTION OF SER^(579)AND ARG^(580)IN β-SUBUNIT OF PENICILLIN G ACYLASE WITH THE METHOD OF SITE-SPECIFIC MUTAGENESIS

作　　者：费俭[1] 林庆聪 蔡国强[1] 张懋弧 黄勤 王应魁 郭礼和[1] 张其玖 

机构地区：[1]中国科学院上海细胞生物学研究所 [2]南京大学生物系 [3]中国科学院北京生物物理研究所

出　　处：《实验生物学报》1992年第3期289-293,共5页Acta Biologiae Experimentalis Sinica

基　　金："863"项目资助

摘　　要：比较青霉素酰化酶(PGA)和青霉素结合蛋白的一级结构,我们推测PGAβ亚基中565～595肽段可能和酶的底物结合功能有关。为此我们将2.6 kb 长的完整的PGA 基因克隆到pTz 18 U 中构建成质粒pTZGA,并用定点突变的技术对Ser^(579)。和Arg^(580)两个氨基酸残基进行了突变研究。在所得到的四种突变子中·Ser^(579)→Gly^(579),Arg^(580)→Gly^(580),Arg^(580)→Glu^(580),Arg^(580)→Lys^(580))Glu^(580)和Gly^(580)没有酶活力,Lys^(580)约有30%的酶活力,Gly^(579)约有70%的酶活力。用ELISA 方法检测了四种突变子和野生型酶蛋白表达量,没有显著差异,这表明Arg^(580)对酶活性有重要意义,是酶活力中心的重要组成部分,可能与催化活性有关。According to the comparison of aminoacid sequence between PGA(Penicillin GAcylase)and PBPs(Penicillin Binding Pro-tein),We suggest that No.565—595 peptidefragment in β-subuint of PGA may be asubstrate-binding site of enzyme.Plasmidp^(TZGA) was constructed by cloning the 2.6kb PGA gene of p^(WOA) into phagemid p^(TZ18U)The technique of site-specific mutagenesiswas used to study the role of residue No.579(Ser)and No.580(Arg)of PGA.Fourkinds of mutants were obtained(Ser^(579)→Gly^(579),Arg^(580)→Gly^(580),Arg^(580)→Glu^(580),Arg^(580)→Lys(580)),both Glu^(580) and Gly^(580)mutants showed no activity of enzyme andLys^(580) mutant remained 30% and Gly^(579)mutant kept 70% activity of wilde type.The same protein expression of fourmutants according to the results of ELISAindicate that mutation does not affect theexpression of PGA,but Arg^(580) residue maybe essential for substrate-binding or cata-lysis of PGA.

关 键 词：青霉素 酰化酶 定点突变 

分 类 号：Q55[生物学—生物化学]