High‐molecular‐weight kininogen binds two molecules of cysteine proteinases with different rate constants

Abstract

Fluorescence titrations showed that high-molecularweight 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 (kas~,l = 23.0 X 106 M -1 s -1 and k~,2 = 3.4 X 106 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~ 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.Key words: Kininogen; Cathepsin; Cystatin; Cysteine proteinase; Kinetics disulfide bond, by limited proteolysis by kallikreins, thereby releasing the kinin segment [2]. The heavy chains of HK and LK are identical, whereas the light chain of HK is substantially larger than that of LK [8].About a decade ago, kininogens were shown to be potent inhibitors of papain-like cysteine proteinases [9,10]. Structural analyses demonstrated that the kininogen heavy chain consists of three domains, designated D1-D3, which share a high internal homology with low-molecular weight cystatins [2,11]. The kininogens therefore were classified as family 3 of the cystatin superfamily [12]. Isolated domains D2 and D3 were shown to inhibit papain-like cysteine proteinases (D2 and D3) and calpain (D2), whereas domain D1 was found to lack inhibitory activity [11,13].We have recently shown that intact human LK simultaneously can bind two molecules of cysteine proteinases with high affinity [14]. In this work we demonstrate that human HK can also bind two molecules of cathepsin S, cruzipain and papain independently. Moreover, kinetic studies of papain binding showed that the two sites bound the proteinase with different rate constants. Materials and methods

The Occluding Loop of Cathepsin B Prevents Its Effective Inhibition by Human Kininogens

“…37 Similarly, HMWK obeys a 2:1 binding stoichiometry, with different rate constants for each binding site. 38 Numerous three-dimensional structures of cystatins and stefins have been described, [39][40][41][42] whereas no structural or mechanistic data are presently available for kininogens.…”

Purification and Biochemical Characterization of a Cystatin-Like Thiol Proteinase Inhibitor from Cicer arietinum (Chickpea)

“…Members of cystatin superfamily also show different binding stoichiometries with papain. The high molecular weight kininogens from human and sheep plasma as well as low molecular weight kininogens from the latter show 1:2 stoichiometry of interaction with papain [72,73].…”

Regulating Cysteine Protease Activity: Essential Role of Protease Inhibitors as Guardians and Regulators

“…Two of these, domains 2 and 3, are able to inhibit a number of cysteine proteases [113], whereas domain 1 can bind calcium [114]. Furthermore, both intact LK and HK can simultaneously bind two molecules of target proteases, but with different affinities [111,112].…”

“…However, by limited proteolysis by kallikreins releasing the kinin segment, they are converted to two-chain forms, consisting of a heavy and a light chain [109]. The heavy chains of HK and LK are identical, whereas the light chain of HK is considerably larger than that of LK [110], resulting in substantial difference in molecular weight: 51 kDa for LK [111] versus 84 kDa for HK [112]. Sequence data have shown that the heavy chains are composed of three tandemly repeated cystatin-like domains, designated domain 1 to domain 3.…”