Liu J N, Tang W, Sun Z Y, Kung W, Pannell R, Sarmientos P, Gurewich V
Vascular Research Laboratory, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA.
Biochemistry. 1996 Nov 12;35(45):14070-6. doi: 10.1021/bi9605393.
Single-chain urokinase-type plasminogen activator or pro-urokinase is a zymogen with an intrinsic catalytic activity which is greater than that of most other zymogens. To study the structural basis for this activity, a three-dimensional homology model was calculated using the crystallographic structure of chymotrypsinogen, and the structure-function relationship was studied using site-directed mutagenesis and kinetic analysis. This model revealed a unique Lys300 in pro-urokinase which could form a weak interaction with Asp355, adjacent to the active site Ser356. It was postulated that this lysine, by its epsilon-amino group, may serve to pull Ser356 close to the active position, thereby inducing the higher intrinsic activity of pro-urokinase. This was consistent with the published finding that a homologous lysine (Lys416) in single chain tissue plasminogen activator when mutated to serine induced some reduction in activity. To test this hypothesis, a site-directed mutant with a neutral residue (Lys300-->Ala) was produced and characterized. The Ala300-pro-urokinase had a 40-fold lower amidolytic activity than that of pro-urokinase. It was also stable in plasma at much higher concentrations than pro-urokinase, reflecting much attenuated plasminogen activation. Plasmin activatability was comparable to that of pro-urokinase, but the resultant two-chain derivative (Ala300-urokinase) had a lower enzymatic activity (approximately 33% that of urokinase) due to a reduction of kcat. Interestingly, the KM of two-chain Ala300-urokinase against plasminogen was 5.8-fold lower than that of urokinase, being similar to that of pro-urokinase which has a KM about 5-fold lower than urokinase. In conclusion, the hypothesis that Lys300 is a key structural determinant of the high intrinsic activity of pro-urokinase was confirmed by these studies. This residue also appears to be important for the full expression of the enzymatic activity of urokinase.
单链尿激酶型纤溶酶原激活剂或尿激酶原是一种具有内在催化活性的酶原，其活性大于大多数其他酶原。为了研究这种活性的结构基础，利用胰凝乳蛋白酶原的晶体结构计算了三维同源模型，并通过定点诱变和动力学分析研究了结构与功能的关系。该模型揭示了尿激酶原中一个独特的赖氨酸300，它可以与活性位点丝氨酸356相邻的天冬氨酸355形成弱相互作用。据推测，这个赖氨酸通过其ε-氨基，可能有助于将丝氨酸356拉近活性位置，从而诱导尿激酶原具有更高的内在活性。这与已发表的研究结果一致，即单链组织型纤溶酶原激活剂中的同源赖氨酸（赖氨酸416）突变为丝氨酸时，活性会有所降低。为了验证这一假设，制备并表征了具有中性残基（赖氨酸300→丙氨酸）的定点突变体。丙氨酸300-尿激酶原的酰胺水解活性比尿激酶原低40倍。它在血浆中的稳定性也比尿激酶原高得多，这反映了纤溶酶原激活作用大大减弱。纤溶酶激活能力与尿激酶原相当，但由于催化常数降低，产生的双链衍生物（丙氨酸300-尿激酶）的酶活性较低（约为尿激酶的33%）。有趣的是，双链丙氨酸300-尿激酶对纤溶酶原的米氏常数比尿激酶低5.8倍，与尿激酶原的米氏常数相似，尿激酶原的米氏常数比尿激酶低约5倍。总之，这些研究证实了赖氨酸300是尿激酶原高内在活性的关键结构决定因素这一假设。这个残基似乎对尿激酶酶活性的充分表达也很重要。
