两种尿激酶原变体(Ala^(175)→Ser,Tyr^(187)→His,Lys^(300)→His和Ala^(175)→Ser,Tyr^(187)→His)的构建及性质研究

CONSTRUCTION AND CHARACTERIZATION OF TWO MUTANTS OF PRO-UROKINASE (Ala^(175)→Ser,Tyr^(187) →His AND Ala^(175) →Ser,Tyr^(187)→His)

利用定点突变方法 ,构建了尿激酶原变体基因muk1(Ala175→Ser,Tyr187→His ,Lys30 0→His)及muk2 (Ala175→Ser ,Tyr187→His) .两种尿激酶原变体在大肠杆菌BL2 1中表达得到包涵体 ,经体外变复性 ,SP Sepharose离子交换柱层析 ,Benzamidine Sepharose吸附除去双链尿激酶 ,得到的蛋白均为银染一条带 .用人工合成的发色底物S2 4 4 4测量muk1、muk2的动力学性质 ,muk1的内在酶活性比野生型 pro UK降低 8倍 ,muk2的内在酶活性比野生型 pro UK降低 2 .5倍 ;它们经纤溶酶 (plasmin)激活后的双链活性与 pro UK相比分别为muk1提高 50 % ,muk2和pro UK相当 .在实验基础上讨论了 pro UK高内源性催化活性的机制及其结构基础 .

Human single chain urokinase type plasminogen activator(scu PA, also named pro UK) is an important thrombolytic agent in therapy of thrombosis. The activation of plasminogen on the surface of fibrin induced by pro UK, is much specific and effective, resulting very small tendency of bleeding from system lytic state. Therefore, great importance has been attached to scu PA in clinics. Though pro UK has some selectivity for fibrin, its higher selectivity for fibrin in human bodies was counteracted by its higher intrinsic activity when it was used in large doses. The risk of bleeding still remains. It was discovered that the stretch of 297～313 amino acids in scu PA formed a loop. Lys 300 ,the only positive charge amino acid in the loop might interact with Asp 355 which was sited near the active center by charge attraction. Thus Ser 365 was pulled , Ser 365 Asp 255 ,His 204 might form the active center on the 3 D structure resulted in the high intrinsic activity of pro UK. Besides, according to the data of crystal structure of chymotrypsin, Asp 194 , His 40 and Ser 32 formed a zymogen triad, which keeps the inactive conformation of chymotrypsin. As a member of the zymogens of serine protease family, pro UK lacks the zymogen triad, His 40 and Ser 32 were replaced respectively by Tyr 187 and Ala 175 . This was also the structure basis of pro UK's high enzymatic activity. To reduce the intrinsic activity of pro UK, 2 mutant genes of pro urokinase, muk1(Ala 175 →Ser, Tyr 187 →His, Lys 300 →His) and muk2(Ala 175 →Ser, Tyr 187 →His) were constructed by site directed mutagenesis, and were expressed in E. Coli BL21. The expressed incusion body was treated by denaturation and renaturation, and purified by SP sepharose ion exchange chromatography and BenzamindineSepharose affinity adsorption. Using the synthetic substrate S2444, the intrinsic activity and the enzymatic activity of two chain form of muk1 and muk2 were measured. The intrinsic activities of muk1 and muk2 were 8 fold and 2.5 fold lower than that of pro urkinase ,respectively. The enzymatic activity of two chain muk1 was 1.5 fold higher than that of urokinase and the activity of muk2 was the same as wild type urokinase. The mechanism and the structure basis of a much higher intrinsic catalytic activity than other zymogens of the serine protease family was discussed.