Abstract
Pro-urokinase has a much higher intrinsic catalytic activity than other zymogens of the serine protease family. Lys300(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 Lys300(c143) to transiently interact with Asp355(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 Lys300(c143) and Asp355(c194). When the charge at Lys300(c143) but not Lys313(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 kcat rather than Km. Some significant structure-function differences between pro-urokinase and its highly homologous counterpart, tissue plasminogen activator, were also found.
Highlights
Pro-urokinase has a much higher intrinsic catalytic activity than other zymogens of the serine protease family
The catalytic reactivity of a two-chain serine protease is primarily determined by the spatial arrangement of the active site residues (His215(c57), Asp260(c102), and Ser356(c195)), formed by a charge interaction between Asp355(c194) and the newly released N-terminal Ile159(c16)
We have recently provided evidence that in single-chain Urokinase-type plasminogen activator (u-PA) or pro-UK, this Asp355(c194) interacts with Lys300(c143), which acts as a surrogate of Ile159(c16) and that this charge interaction explains the relatively high intrinsic catalytic activity (ICA) of pro-UK [13]
Summary
Pro-urokinase has a much higher intrinsic catalytic activity than other zymogens of the serine protease family. UK is a typical enzyme of this family, pro-UK has several unusual properties that distinguish it from other serine protease zymogens These include its high intrinsic catalytic activity (ICA) [7,8,9], its reversible inhibition by diisopropylfluorophosphate [10], the existence of a unique hypercatalytic transitional state against plasminogen during the conversion or pro-UK to UK [11], and its strong fibrin promotion, which gives the ICA of pro-UK an activity equivalent to the TCA of UK [12]. It was noted that the Lys300(c143) residue was located in a loop region [297–313] believed to be “flexible” because of its very high isotropic temperature factor in the homologous structures of trypsinogen and chymotrypsinogen [13] The function of this “flexible loop” in serine proteases is unknown, it is a part of the activation domain important in trypsinogen activation [14]. The effect of mutations in this region on the TCA is unknown, but the
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