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High‐molecular‐weight kininogen binds two molecules of cysteine proteinases with different rate constants
Author(s) -
Turk Boris,
Stoka Veronika,
Turk Vito,
Johansson Gunnar,
Cazzulo Juan Jose,
Björk Ingemar
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00611-4
Subject(s) - high molecular weight kininogen , chemistry , kininogen , papain , sedimentation equilibrium , binding constant , binding site , equilibrium constant , stereochemistry , enzyme , biochemistry , kallikrein , inorganic chemistry
Fluorescence titrations showed that high‐molecular‐weight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped‐flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants ( k ass,1 = 23.0 × 10 6 M − s −1 and k ass2 , = 3.4 × 10 6 M −1 s −1 . As for low‐molecular‐weight kininogen, comparison of these kinetic constants with previous data for intact low‐ and high‐molecular‐weight kininogen and the separated domains indicated that the faster‐binding site is also the tighter‐binding site and is that of domain 3, whereas the slower‐binding, lower‐affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M r of high‐molecular‐weight kininogen was determined to be 83 500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.