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High‐affinity binding of two molecules of cysteine proteinases to low‐molecular‐weight kininogen
Author(s) -
Turk Boris,
Stoka Veronika,
Björk Ingemar,
Boudier Christian,
Johansson Gunnar,
Dolenc Iztok,
Colic Adrijana,
Bieth Joseph G.,
Turk Vito
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040922
Subject(s) - chemistry , cysteine , kininogen , biochemistry , high molecular weight kininogen , enzyme , kallikrein
Human low‐molecular‐weight kininogen (LK) was shown by fluorescence titration to bind two molecules of ca‐thepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2 :1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants k ass, I = 10.7‐24.5 × 10 6 M −1 s −1 and k ass, 2 = 0.83‐1.4 × 10 6 M −1 s −1 . Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster‐binding site is also the tighter‐binding site and is present on domain 3, whereas the slower‐binding, lower‐affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain.