COMPLEXATION OF THE TISSUE-PLASMINOGEN ACTIVATOR PROTEASE WITH BENZAMIDINE-TYPE INHIBITORS - INTERFERENCE BY THE KRINGLE-2 MODULE

Well-resolved high-field H-1 NMR signals between -0.1 and -0.7 ppm aff ord convenient probes to monitor the conformational state of the tissu e plasminogen activator (tPA) protease, modulated by covalent inhibito r binding or activation cleavage [Hu. C.-K., Kohnert, U., Wilhelm, O., Fischer, S., & Llinas, M. (1994) Biochemistry 33, 11760-11766]. We ha ve investigated recombinant BM 06.022 (a domain-deletion variant mutan t from Escherichia coli comprising the kringle 2 and protease modules) and protease constructs of tPA in both single-chain (sc) and two-chai n (tc) forms, The two proteins were studied when confronted with the n oncovalent (i.e., reversible) active site inhibitors benzamidine and a series of bisbenzamidine derivatives: 2,5-bis(4-amidinobenzylidene)cy clopentanone, 2,6-bis(4-amidinobenzylidene)cyclohexanone, 2,7-bis(4-am idinobenzylidene)cycloheptanone, and 2,8-bis(4-amidinobenzylidene)cycl ooctanone. At pH 4.6, the H-1 NMR spectrum is sensitive to complexati on of the protease module with the various effecters. The amplitude of the inhibitor-shifted resonances is more pronounced for the tc-protea se than for the sc-protease, suggesting that access of inhibitors to t he protease catalytic site is facilitated upon conversion to the tc fo rm, The effects detected by the NMR spectrum suggest a biphasic proces s, involving stronger (primary) and weaker (secondary) bindings to a s ingle protease active site. Binding to the protease module in tc-BM 06 .022 essentially generates the same spectral characteristics as detect ed upon binding to the isolated tc-protease construct, In contrast, a negligible perturbation by the inhibitors is observed on the (sc) BM 0 6.022, Hence, in the intact BM 06.022 the kringle 2 is structurally co upled to the protease module thus interfering with inhibitor molecules from accessing the protease active site. These domain-domain interact ions relax upon conversion to the catalytically active re form, thus d ecoupling the kringle 2 from the protease module in BM 06.022 while si multaneously exposing the active site to become accessible to effecter s or substrates.