单链尿激酶型纤溶酶原激活剂Kringle结构域催化功能探讨

Catalysis-related Function of Kringle Domain of Single Chain Urokinase-type Plasminogen Activator

Kringle(K)结构域广泛存在于与凝血和纤溶相关的各种因子中 .虽然 K结构域在一级结构上是一种比较保守的结构域 (与其他结构域相比 ) ,但是 K结构域的功能在各种因子中的作用有很大的区别 .为探讨单链尿激酶型纤溶酶原激活剂 (scu- PA)的 K结构域功能 ,构建了在 scu- PA的 K结构域的 1 1 8位 Gly与 1 1 9位 Leu之间插入 PRGDWR序列的突变体 (称为 insert mutant B,In B) ,并测定了野生型 scu- PA与 In B的纤溶相关反应的动力学常数 . scu- PA与 In B水解 S- 2 4 4 4反应的 Km 值无明显变化 (分别为 60 .4与 56.8μmol·L-1) ,而 In B的 kcat值 (0 .33s-1)比 scu- PA的 kcat值 (7.31 s-1)降低很多 ;In B激活纤溶酶原反应的 Km 值 (0 .397μmol· L-1)比 scu- PA Km 值(0 .648μmol·L-1)降低 40 % ,但 kcat值 (0 .0 1 65s-1)比 scu- PA的 kcat值 (0 .0 62 6s-1)降低 74% .以上结果说明 :K结构域主要与反应活性相关 ,而与酶及底物的亲和性无关 .

Kringle domain is a kind of conservative domain that exists in many thrombus forming and thrombolysis related factors.The primary structure of kringle domain is high conserved(compared with other domains in those factors).To study the function of Kringle domain of single chain urokinase type plasminogen activator(scu PA),an Kringle domain inserted mutant of scu PA was constructed by inserting PRGDWR peptide at the site between Gly 118 and Leu 119 (Insert mutant B,InB),and the kinetic constants of thrombolytic related reactions were detected. K m value of hydrolytic reaction of S 2444 catalyzed by the two molecule was not significantly different(60.4 and 56.8 μmol·L -1 respectively),but the k cat value of InB(0.33 s -1 )was decreased a lot compared with the k cat of scu PA(7.31 s -1 ).The K m value of InB(0.397 μmol·L -1 )of the activation reaction of plasminogen was 40% lower than the K m value of scu PA(0.648 μmol·L -1 ),but the k cat value of InB(0.0165 s -1 )was over 70% lower than that of scu PA(0.0626 s -1 ).These results suggest that Kringle domain of scu PA is related with the catalytic activity of scu PA,but has no influence on the affinity between scu PA and its substrate.