Pro-urokinase has a much higher intrinsic catalytic activity than other zymogens of the serine protease family. Lys 300(c143) in an apparent "flexible loop" region (297-313) was previously shown to be an important determinant of this intrinsic catalytic activity. This was related to the loop allowing the positive charge of Lys 300(c143) to transiently interact with Asp 355(c194) , thereby inducing an active conformation of the protease domain (Liu, J. N., Tang, W., Sun, Z., Kung, W., Pannell, R., Sarmientos, P., and Gurewich, V. (1996) Biochemistry 35, 14070 -14076). To further test this hypothesis, the charge at position 300(c143) and the flexibility of the loop were altered using site-directed mutagenesis designed according to a computer model to affect the interaction between Lys 300(c143) and Asp 355(c194). When the charge at Lys 300(c143) but not Lys 313(c156) was reduced, a significant reduction in the intrinsic catalytic activity occurred. Similarly, when the flexibility (wobbliness) of the loop was enhanced reducing the size of side chain, the intrinsic catalytic activity was also reduced. By contrast, when the loop was made less flexible, the intrinsic catalytic activity was increased. These findings were consistent with the hypothesis. The effects of these mutations on two-chain activity were less and often discordant with the intrinsic catalytic activity, indicating that they can be modulated independently. This structure-function disparity can be exploited to create a more zymogenic pro-urokinase (lower intrinsic catalytic activity) with a high catalytic activity, as exemplified by two of the mutants. The changes in intrinsic catalytic activity and two-chain activity induced by the mutations were due to changes in k cat rather than K m . Some significant structure-function differences between prourokinase and its highly homologous counterpart, tissue plasminogen activator, were also found.

Urokinase-type plasminogen activator (u-PA) or its amino-terminal fragment (ATF) containing the u-PA receptor (u-PAR) binding domain, is known to promote monocyte adhesion. In the present study, U937 monocyte adhesion to a plastic surface was used to investigate the mechanism of its promotion by u-PA and ATF. Adhesion was found to be inhibited by cycloheximide or actinomycin D, implicating protein synthesis and gene expression in u-PA-induced monocyte adhesion. Adhesion was prevented by 2-deoxyadenosine 3-monophosphate, indicating that a cAMP-dependent pathway of signal transduction was involved. This concept was supported by the complementary finding that u-PA-induced adhesion was greatly promoted by forskolin, cholera toxin, or 8-bromo-cAMP, which by themselves induced little adhesion. Furthermore, similar to many other cAMP-dependent activities, cGMP diminished u-PA-induced adhesion. When u-PA or ATF was treated with immobilized carboxypeptidase B, its proadhesive effect was abolished, implicating the involvement of carboxylterminal lysine residues (Lys 158 on u-PA and Lys 135 on ATF). Moreover, when a carboxyl-terminal lysine analog was added, the proadhesive effect of carboxypeptidase B-treated u-PA or ATF was restored. In conclusion, the present study indicates that u-PA-or ATF-induced monocyte adhesion involves cAMP-dependent signal transduction, which is triggered by u-PAR binding. It is also critically dependent on the presence of a carboxylterminal lysine.

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