Open AccessEnzymatic properties of single‐chain and two‐chain forms of a Lys 158 → Glu 158 mutant of urokinase–type plasminogen activator
Author(s) -
LIJNEN H. Roger,
HOEF Berthe,
NELLES Luc,
HOLMES William E.,
COLLEN Désiré
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb13871.x
Subject(s) - plasmin , urokinase , plasminogen activator , chemistry , biochemistry , enzyme , peptide , microbiology and biotechnology , sepharose , site directed mutagenesis , mutant , stereochemistry , biology , genetics , endocrinology , gene
Recombinant single‐chain urokinase‐type plasminogen activator (rscu‐PA), in which the plasmin‐sensitive peptide bond Lys 158 ‐Ile 159 is destroyed by site‐specific mutagenesis of LYS 158 to Glu (rscu‐PA‐Glu 158 ), is quantitatively converted to two‐chain urokinase‐type plasminogen activator (rtcu‐PA‐Glu 158 ) by treatment with endoproteinase Glu‐C ( Staphylococcus aureus V8 proteinase). The catalytic efficiency ( k 2 /K m ) of rscu‐PA‐Glu 158 for the activation of plasminogen is 20 times lower (0.0001 μM −1 s −l ) than that of rscu‐PA (0.002 μM −1 s −1 ). In contrast, rtcu‐PA‐Glu 158 has very similar properties to rtcu‐PA obtained by digestion of rscu‐PA with plasmin, including binding to benzamidine‐Sepharose, catalytic efficiency for the activation of plasminogen (0.035 μM −1 s −1 versus 0.046 μM −1 s −1 ) and fibrinolytic activity in an in vitro plasma clot lysis system. It is concluded that the amino acid in position 158 is a main determinant of the functional properties of single‐chain urokinase‐type plasminogen activator but not of the two‐chain form.