title: Complexation of the tissue plasminogen activator protease with benzamidine-type inhibitors: interference by the kringle 2 module.
abstract: Well-resolved high-field 1H NMR signals between -0.1 and -0.7 ppm afford convenient probes to monitor the conformational state of the tissue plasminogen activator (tPA) protease, modulated by covalent inhibitor binding or activation cleavage [Hu, C.-K., Kohnert, U., Wilhelm, O., Fischer, S., & Llinas, M. (1994) Biochemistry 33, 11760-11766]. We have investigated recombinant BM 06.022 (a domain-deletion variant mutant from Escherichia coli comprising the kringle 2 and protease modules) and protease constructs of tPA in both single-chain (sc) and two-chain (tc) forms. The two proteins were studied when confronted with the noncovalent (i.e., reversible) active site inhibitors benzamidine and a series of bisbenzamidine derivatives: 2,5-bis(4-amidinobenzylidene)cyclopentanone, 2,6-bis(4-amidinobenzylidene)cyclohexanone, 2,7-bis(4-amidinobenzylidene)cycloheptanone, and 2,8-bis(4-amidino- benzylidene)cyclooctanone. At pH* 4.6, the 1H NMR spectrum is sensitive to complexation of the protease module with the various effectors. The amplitude of the inhibitor-shifted resonances is more pronounced for the tc-protease than for the sc-protease, suggesting that access of inhibitors to the protease catalytic site is facilitated upon conversion to the tc form. The effects detected by the NMR spectrum suggest a biphasic process, involving stronger (primary) and weaker (secondary) bindings to a single protease active site. Binding to the protease module in tc-BM 06.022 essentially generates the same spectral characteristics as detected upon binding to the isolated tc-protease construct. In contrast, a negligible perturbation by the inhibitors is observed on the (sc) BM 06.022. Hence, in the intact BM 06.022 the kringle 2 is structurally coupled to the protease module thus interfering with inhibitor molecules from accessing the protease active site. These domain-domain interactions relax upon conversion to the catalytically active tc form, thus decoupling the kringle 2 from the protease module in BM 06.022 while simultaneously exposing the active site to become accessible to effectors or substrates.
authors.affiliation: Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: C K
authors.initials: CK
authors.lastname: Hu
authors.forename: U
authors.initials: U
authors.lastname: Kohnert
authors.forename: J
authors.initials: J
authors.lastname: Sturzebecher
authors.forename: S
authors.initials: S
authors.lastname: Fischer
authors.forename: M
authors.initials: M
authors.lastname: Llinas
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D001550:Benzamidines / Q000737:chemistry / Q000378:metabolism*; D003619:Dansyl Compounds / Q000378:metabolism; D006801:Humans; D018082:Kringles / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011480:Protease Inhibitors / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D001550:Benzamidines; D003619:Dansyl Compounds; D011480:Protease Inhibitors; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; D010959:Tissue Plasminogen Activator