A site-directed mutagenesis of pro-urokinase which substantially reduces its intrinsic activity

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 Lys(300) in pro-urokinase which could form a weak interaction with Asp(355), adjacent to the active site Ser(356). It was postulated that this lysine, by its epsilon-amino group, may serve to pull Ser(356) 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 (Lys(416)) 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 (Lys(300) --> Ala) was produced and characterized. The Ala(300)-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 (Ala(300)-urokinase) had a lower enzymatic activity (approximate to 33% that of urokinase) due to a reduction of k(cat). Interestingly, the K-M Of two-chain Ala(300)-urokinase against plasminogen was 5.8-fold lower than that of urokinase, being similar to that of pro-urokinase which has a K-M about 5-fold lower than urokinase. In conclusion, the hypothesis that Lys(300) 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.