尿激酶原变体Glu151-Glu154-mscu-PA的构建及其动力学性质

Construction of Pro urokinase Mutant Glu151-Glu154-mscu-PA and Characterization of its Kinetic Properties

利用寡核苷酸介导的定点突变方法 ,将重组人尿激酶原 ( recombinant single chain uroki-nase- type plasminogen activator,rscu- PA)中 1 51位赖氨酸 ( Lys1 51 )突变为谷氨酸 ( Glu1 51 ) ,1 54位精氨酸 ( Arg1 54)突变为谷氨酸 ( Glu1 54) ,得到尿激酶原变体基因 ( mscu- PA) .尿激酶原变体和未突变的重组尿激酶原均在 E.coli中获得表达 ,超声后所得包涵体经体外变复性并得到纯化 .结果表明 ,尿激酶原变体对纤溶酶 ( plasmin)的敏感性比未突变的重组尿激酶原低约 40 % ,转变纤溶酶原 ( Glu- plasminogen)为纤溶酶的活性基本相同 .两种产物经纤溶酶活化后 ,分别得到了双链尿激酶 ( rtcu- PA)和双链尿激酶变体 ( mtcu- PA) .它们对人工合成的发色底物 S2 4 4 4反应的动力学基本一致 ,对 Glu- plasminogen的催化反应的米氏动力学常数 Km 基本一致 ,但 mtcu- PA的 Kcat仅为rtcu- PA的 80 % .酪蛋白降解系统 ( caseinolytic system)实验表明 ,在纤维蛋白和纤溶酶原存在的情况下 ,尿激酶原变体较未突变尿激酶原能加快酪蛋白的降解 ,说明 mtcu-

rscu PA and its mutant constructed in vitro by site directed mutagenesis of Lys151,Arg154 in rscu PA to Glu151,Glu154(mscu PA) were both expressed in Escherichia coli .After in vitro denaturation and renaturation,the rscu PA and mscu PA were purified to homogeneity by Zn 2+ selective precipitation,anti u PA IgG Sepharose CL 4B affinity chromatography.The activation by plasmin of mscu PA was 40% lower than that of rscu PA.mscu PA and rscu PA were found essentially identical in Glu plasminogen activation.After activation by plasmin,the kinetic constants for the resultant mtcu PA against synthetic substrate S2444 hydrolysis were found about 90% of rtcu PA.Although the K m of two chain mtcu PA against Glu plasminogen was similar to that of rtcu PA,mtcu PA had a lower enzymatic activity(about 80% that of rtcu PA) due to a reduction of K cat .In caseinolytic system,with the fibrin and plasminogen,mscu PA could speed up the decomposition of casein more than rscu PA,which suggested that mtcu PA had some fibrin specificity.