Kinetics of inhibition of bovine cathepsin S by bovine stefin B

Turk, Boris; Čolič, Adrijana; Stoka, Veronika; Türk, Vito

doi:10.1016/0014-5793(94)80405-2

Cited by 34 publications

(32 citation statements)

References 33 publications

(28 reference statements)

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“…A linear dependence of the observed pseudo first-order rate constant on inhibitor concentration was observed for all enzyme-inhibitor pairs investigated (Fig. 4), consistent with a simple, competitive inhibition mechanism [8,19]. Apparent second-order association rate constants, kass, were obtained from the slopes of these plots.…”

Section: Inhibition Kineticssupporting

confidence: 68%

Identification of bovine stefin A, a novel protein inhibitor of cysteine proteinases

et al. 1995

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For the first time, three different stefins, A, B and C, have been isolated from a single species. The complete amino acid sequence of bovine stefin A was determined. The inhibitor, with a calculated Mr of 11,123, consists of 98 amino acid residues. Although it exhibits considerable similarity to human and rat stefin A, some significant differences in inhibition kinetics were found. Bovine stefin A bound tightly and rapidly to cathepsin L (ka~ = 9.6.106 M-~-s -t, Kj = 29 pM). The binding to cathepsin H was also rapid (ka~ = 2.1 "106 M -I "s-J), but weaker (K i = 0.4 nM) due to a higher dissociation rate constant. In contrast, the binding to cathepsin B was much slower (kas ~ = 1.4. l0 s M -I" s-~), hut still tight (Ks = 1.9 nM).Key words: Stefin; Cystatin; Cysteine proteinase; Cathepsin; Amino acid sequence tures of chicken cystatin [9] and stefin B in complex with papain [10], a novel enzyme-inhibitor binding mechanism was proposed. Three regions of the inhibitor were shown to interact with the enzyme: two hairpin loops and the N-terminal part [9,10], which appears to be more important in the cystatins [11][12][13].In this paper we describe the purification and characterization of stefin A from bovine skin. The complete amino acid sequence of the inhibitor was determined and compared with other known sequences of inhibitors from the stefin family. Moreover, the kinetics of interaction of the isolated inhibitor with mammalian cysteine proteinases were characterized to clarify further the role of cystatins as potent physiological regulators of cysteine proteinase activity. Materials and methods

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Section: Inhibition Kineticssupporting

confidence: 68%

Identification of bovine stefin A, a novel protein inhibitor of cysteine proteinases

et al. 1995

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“…Only 10-12% of the activity was lost in 1 hour at pH 7.5 (Kirschke et al, 1989;Brömme et al, 1993). Cystatins and stefins are potent inhibitors of cathepsin S (Brömme et al, 1991;Turk et al, 1994).…”

Section: (A) Cathepsin S (I) Properties and Tissue Distributionmentioning

confidence: 99%

Cysteine Peptidases of Mammals: Their Biological Roles and Potential Effects in the Oral Cavity and Other Tissues in Health and Disease

Dickinson

2002

Critical Reviews in Oral Biology & Medicine

158

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Cysteine peptidases (CPs) are phylogenetically ubiquitous enzymes that can be classified into clans of evolutionarily independent proteins based on the structural organization of the active site. In mammals, two of the major clans represented in the genome are: the CA clan, whose members share a structure and evolutionary history with papain; and the CD clan, which includes the legumains and caspases. This review focuses on the properties of these enzymes, with an emphasis on their potential roles in the oral cavity. The human genome encodes at least (but possibly no more than) 11 distinct enzymes, called cathepsins, that are members of the papain family C1A. Ten of these are present in rodents, which also carry additional genes encoding other cathepsins and cathepsin-like proteins. Human cathepsins are best known from the ubiquitously expressed lysosomal cathepsins B, H, and L, and dipeptidyl peptidase I (DPP I), which until recently were considered to mediate primarily "housekeeping" functions in the cell. However, mutations in DPP I have now been shown to underlie Papillon-Lefèvre syndrome and pre-pubertal periodontitis. Other cathepsins are involved in tissue-specific functions such as bone remodeling, but relatively little is known about the functions of several recently discovered enzymes. Collectively, CPs participate in multiple host systems that are active in health and in disease. They are involved in tissue remodeling and turnover of the extracellular matrix, immune system function, and modulation and alteration of cell function. Intracellularly, CPs function in diverse processes including normal protein turnover, antigen and proprotein processing, and apoptosis. Extracellularly, they can contribute directly to the degradation of foreign proteins and the extracellular matrix. However, CPs can also participate in proteolytic cascades that amplify the degradative capacity, potentially leading to pathological damage, and facilitating the penetration of tissues by cancer cells. We know relatively little regarding the role of human CPs in the oral cavity in health or disease. Most studies to date have focused on the potential use of the lysosomal enzymes as markers for periodontal disease activity. Human saliva contains high levels of cystatins, which are potent CP inhibitors. Although these proteins are presumed to serve a protective function, their in vivo targets are unknown, and it remains to be discovered whether they serve to control any human CP activity.

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“…The experiments were carried out under pseudo-first-order conditions with a 10-fold molar ratio of HK to papain, which precluded determination of the association rate constant for the slower-binding site (see above). All progress curves had a final slope of zero, showing that the reactions were essentially irreversible at the inhibitor concentrations used, and were thus fitted to a simple exponential function [31]. The plot of the observed pseudo-firstorder rate constant vs. HK concentration was linear and gave an association rate constant of 33.3 + 1.1 x 106 M -1 s -1 after correction for substrate competition.…”

Section: Kinetics Of Bindingmentioning

confidence: 99%

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

et al. 1996

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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

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Kinetics of inhibition of bovine cathepsin S by bovine stefin B

Cited by 34 publications

References 33 publications

Identification of bovine stefin A, a novel protein inhibitor of cysteine proteinases

Identification of bovine stefin A, a novel protein inhibitor of cysteine proteinases

Cysteine Peptidases of Mammals: Their Biological Roles and Potential Effects in the Oral Cavity and Other Tissues in Health and Disease

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

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