title: Inactivation of human high- and low-molecular-weight urokinases. Analysis of their active site.
abstract: We previously demonstrated that 3,4-dihydro-3,4-dibromo-6-bromomethylcoumarin (dihydrocoumarin I) inhibited high-molecular-weight urokinase through a mechanism-based (suicide) inactivation (M. Reboud-Ravaux, G. Desvages and F. Chapeville (1982) FEBS Lett. 140, 58-62). In order to define the site of alkylation, peptic peptides were prepared from urokinase (heavy form) treated first by tritiated dihydrocoumarin I. After separation by reverse-phase HPLC, the labelled fragments were sequenced. His-46 in the B-chain of urokinase (heavy form) had been selectively alkylated, proving that this amino acid forms part of the active site. 3,4-Dihydro-3-benzyl-6-chloromethylcoumarin (dihydrocoumarin II) was more reactive than dihydrocoumarin I against urokinase (heavy form) by a factor of 130. Low-molecular-weight urokinase was inactivated by dihydrocoumarin II slightly more slowly than urokinase (heavy form), showing a decrease of 30% in the corresponding second-order rate constant. In contrast, dihydrocoumarin I displayed an analogous reactivity against light and heavy forms of urokinase. As expected, in the absence of the alkylating moiety, such as in 3,4-dihydro-3-benzylcoumarin (dihydrocoumarin III), no inactivation was observed. It is note-worthy that dihydrocoumarin II which carried an extra-aromatic group fitted well within the active site of light and heavy urokinases, suggesting a nonpolar character for their primary binding site.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003374:Coumarins / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000652:urine; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D014316:Tritium; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000652:urine
chemical_list: D000596:Amino Acids; D003374:Coumarins; D007527:Isoenzymes; D014316:Tritium; C034805:3,4-dihydro-3,4-dibromo-6-bromomethylcoumarin; D014568:Urokinase-Type Plasminogen Activator