Kinetics of binding of chicken cystatin to papain

Björk, Ingemar; Alriksson, E; Ylinenjärvi, K

doi:10.1021/bi00430a022

Cited by 69 publications

(109 citation statements)

References 29 publications

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“…All variants inhibit papain as well as cathepsin B and L, but weaker than chicken cystatin. The general interaction model of chicken cystatin with papain [7,8,13] was confirmed experimentally: as predicted, both hairpin loops as well as the N-terminal trunk of the inhibitor contribute to complex formation. However, the contributions of the three binding regions to the free energy change during complex formation seem to be not simply additive.…”

Section: Discussionsupporting

confidence: 56%

“…According to the so-called 'elephant-trunk model', the enzyme-inhibitor complex is formed mainly by hydrophobic interactions between chicken cystatin and the complementary active-site cleft of papain, whereby cystatin contributes the N-terminal 'trunk' (Leu7-Gly9), the first hairpin loop (Gln53-Gly57) and the second hairpin loop (Pro]°3-Leul°5). The validity of this model was confirmed by elucidation of the structure of the complex formed between S-carboxymethylated papain and stefin B [15] as well as by kinetic data obtained for the interactions of native and/or modified cystatins with different cysteine proteinases [7,8,12,16].…”

Section: ~ Introductionmentioning

confidence: 77%

“…E-64, t.-3-carboxy-2,3-trans-epoxy-propionyl-leucyl~Lmido-(4-guanidino)butane; NH-Mec, 7-(4-methyl) coumaryl-amide; .5, benzyloxycarbonyl; rCC, AEF-[S1M, M29I, M89L] chicken cysatin, (recombinant chicken cystatin); AV55, AEF-[S1 M, M29I, AV55, VI89L] chicken cystatin; AV55-$56, AEF-[S1M, M29I, AV55, AS56, VI89L] chicken cystatin; loop2-KD2, AEF-[S 1 M, M29I, M89L, S 101 I, 1102D, P103I, Wl04Q, L105, N106R, Q107I, I108A] chicken cystatin; 1P103-L105, AEF-[S1M, M29I, M89L, AP103, AWl04, ALl05,] chicken cystatin; AI 102-Q 107, AEF-[S 1 M, M29I, M89L, AI 102, AP 103, AWl04, ALl05, AN106, AQ107] chicken cystatin; N-del 2, [AS1-P11, V12A, PI3E, V14F, DI5M, M29I, M89L] chicken cystatin. [4][5][6][7][8][9]. Synthetic genes of chicken cystatin and variants of them have been expressed and characterized [10][11][12].…”

Section: ~ Introductionmentioning

confidence: 99%

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Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

et al. 1995

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~. IntroductionChicken cystatin, a small protein-type inhibitor of cysteine proteinases, represents a well studied member of the cystatin ,uperfamily (for reviews see [2,3]). It was shown to be a reversble tight-binding inhibitor of papain and papain-like enzymes 4Corresponding author. Fax: (49) (89) 5160 4735. E-mail: tbbreviations. E-64, t.-3-carboxy-2,3-trans-epoxy-propionyl-leucyl~Lmido-(4-guanidino)butane; NH-Mec, 7-(4-methyl) coumaryl-amide; . [4-9]. Synthetic genes of chicken cystatin and variants of them have been expressed and characterized [10][11][12]. Crystal and solution structures of native and recombinant chicken cystatin are known [13,14] and a model of the interaction of chicken cystatin with papain has been proposed [7,13]. According to the so-called 'elephant-trunk model', the enzyme-inhibitor complex is formed mainly by hydrophobic interactions between chicken cystatin and the complementary active-site cleft of papain, whereby cystatin contributes the N-terminal 'trunk' (Leu7-Gly9), the first hairpin loop (Gln53-Gly57) and the second hairpin loop (Pro]°3-Leul°5). The validity of this model was confirmed by elucidation of the structure of the complex formed between S-carboxymethylated papain and stefin B [15] as well as by kinetic data obtained for the interactions of native and/or modified cystatins with different cysteine proteinases [7,8,12,16].Detailed analyses of the N-terminal segments of chicken cystatin and human cystatin C indicate a substrate-like interaction of this region with residues of the subsites $3 and $2 of the proteinase. Especially the amino acid residues preceding the conserved Gly 9 of chicken cystatin or Gly ~1 of human cystatin C have distinct but different effects on the binding to papain, actinidin, ficin, cathepsin B or cathepsin L [17 20].The contribution of the first hairpin loop of cystatins to complex formation has not yet been investigated in such detail. From studies with KVVAG-and QVTAG-mutants of cystatin A, Nikawa et al. [21] proposed that the first hairpin loop is not essential for cysteine proteinase inhibition. But the importance of this region is strongly suggested by (i) the structural data of the crystallized stefin B-papain complex [15], (ii) its conserved primary structure (QxVxG-region) [2] and (iii) inhibition properties measured with substitution variants of oryzacystatin [22] and chicken cystatin [12]. The latter data provide first evidence that amino acid residues of this region are partially responsible for the distinct affinities of cystatins for different cysteine proteinases. However, no data have been presented so far on variants of the first hairpin loop missing conserved amino acid residues.Little is known about the importance of the second hairpin loop for the stability of the molecule itself or its contribution to complex formation. Using spectroscopic methods, Lindahl and co-workers [6] demonstrated that Trpl°4 of chicken cystatin must be involved in the interaction with papain. These early data...

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Section: Discussionsupporting

confidence: 56%

Section: ~ Introductionmentioning

confidence: 77%

Section: ~ Introductionmentioning

confidence: 99%

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Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

et al. 1995

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“…Amino acid analyses were carried out on an LKB 4151 Alpha Plus amino acid analyser (Pharmacia LKB) after hydrolysis of the samples in 6 M-HCI at 110°C for 24 and 72 h. N-Terminal sequences were analysed as described previously (Bjork et al, 1989a).…”

Section: Protein Analysesmentioning

confidence: 99%

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Björk

Ylinenjärvi

Olson

et al. 1992

Biochemical Journal

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Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proporti6n of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.

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“…The crystal structure of chicken cystatin [6,7] suggests that the proteinase binding site comprises three regions of the polypeptide chain, which together form a wedge-shaped edge of the molecule that fits into the active-site cleft of papain with minimal conformational changes of either protein. The spectroscopic and kinetic characteristics of the interactions of chicken cystatin and cystatin C with those target proteinases that have been studied so far are highly similar [2, 5,8], indicating that the two inhibitors bind to the enzymes in a closely related manner.…”

Section: Introductionmentioning

confidence: 99%

Biphasic transition curve on denaturation of chicken cystatin by guanidinium chloride Evidence for an independently unfolding structural region

Björk

Pol

1992

FEBS Letters

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FaPultrav~let circular diohroism and tryptophan fluorescence measurements showed that the reversible unfolding of the c3,steine proteinase inhibitor, chicken eystatin, by guanidinium chloride is a two-step process with transition midpoints at ..-3.4 and ~5.4 M denaturant. The partially unfolded intermediate had both far-and near-ultraviolet circular diehroism and fluorescence emission spectra comparable to those of the native protein. The largely retained tertiary structure suggests that the intermediate represents a species in which a separate region of lower stability has been unfolded, rather than an intermediate of the 'molten globule' type. Such a structurally independent region is apparent in the three-dimensional structure of the inhibitor.

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Kinetics of binding of chicken cystatin to papain

Cited by 69 publications

References 29 publications

Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Biphasic transition curve on denaturation of chicken cystatin by guanidinium chloride Evidence for an independently unfolding structural region

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