Mapping of the catalytic site of CHO-t-PA and the t-PA variant BM 06.022 by synthetic inhibitors and substrates.
BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X-ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA. Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.
Stürzebecher
J
J
Institute of Pharmacology & Toxicology, Medical Academy Erfurt, Germany.
Neumann
U
U
Kohnert
U
U
Kresse
G B
GB
Fischer
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001550:Benzamidines / Q000494:pharmacology; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D001550:Benzamidines; D009842:Oligopeptides; D011994:Recombinant Proteins; D014357:Trypsin; D013917:Thrombin; D010959:Tissue Plasminogen Activator