Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes

Björk, Ingemar; Pol, Ewa; Raub-Segall, Elke; Abrahamson, Magnus; Rowan, Andrew D.; Mort, John S.

doi:10.1042/bj2990219

Cited by 84 publications

(116 citation statements)

References 30 publications

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“…The crystal structure of human cathepsin B shows an additional loop, not present in papain, of about 20 amino acid residues, which partially occludes the active site. This loop was proposed to interfere with inhibitor binding [25], a suggestion supported by observations that the association rate constants for the interactions of inhibitors with cathepsin B are markedly lower than those for the binding to other enzymes [11,12,23].…”

Section: Discussionmentioning

confidence: 84%

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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: Discussionmentioning

confidence: 84%

“…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].…”

mentioning

confidence: 99%

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

et al. 1995

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“…A structural element that has been shown to be at least partly responsible for the target enzyme specificity of cystatin C and of crucial importance for efficient cystatin C binding of cathepsin B is the N-terminal segment with ArgLeu-Val-Gly at positions 8 -11 (40,53). Leu-9 and Val-10, interacting with target enzyme substrate subpockets S 3 and S 2 , respectively, are especially important for a fast interaction between the cystatin and cathepsin B (54,55). The corresponding N-terminal segment in cystatin F is Val-Lys-Pro-Gly.…”

Section: Resultsmentioning

confidence: 99%

“…For fractionation of blood plasma, solid ammonium sulfate was added to a series of tubes containing 2-ml portions of plasma to achieve 25,30,40,45,50,55,60,70, and 75% (w/v) saturation. Following overnight incubation at 4°C, the tubes were centrifuged (10,000 ϫ g, 30 min), the supernatants were collected, and the precipitates were dissolved in water.…”

Section: Methodsmentioning

confidence: 99%

Cystatin F Is a Glycosylated Human Low Molecular Weight Cysteine Proteinase Inhibitor

Ni¹,

Fernandez²,

Danielsson³

et al. 1998

Journal of Biological Chemistry

144

126

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“…The importance of the N-terminal region for the typical tight enzyme-binding property of cystatin C has previously been established [1,3,4]. We have shown that Leu-9 is the most discriminating residue for binding to cathepsins B, H, L and S, and thereby also a major determinant for the inhibitor's natural inhibition profile.…”

Section: Introductionmentioning

confidence: 92%

Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases

Mason

Sol‐Church

Abrahamson

1998

Biochemical Journal

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We used site-directed mutagenesis to alter the specificity of human cystatin C, an inhibitor with a broad reactivity against cysteine proteinases. Nine cystatin C variants containing amino acid substitutions in the N-terminal (L9W, V10W, V10F and V10R) and/or the C-terminal (W106G) enzyme-binding regions were designed and produced in Escherichia coli. It was discovered that the inhibition profile of the cystatin could be altered by changing residues 9 and 10, which are proposed to bind in the S3 and S2 substrate-binding pockets respectively of the enzymes. All of the variants with substitutions in the N-terminal segment displayed decreased binding to cathepsins B and H, indicating that the S3 and S2 pockets of these enzymes cannot easily accommodate large aromatic residues. The introduction of a charged residue into S2 (variant V10R) created a more specific inhibitor to distinguish cathepsin B from cathepsin H. Cathepsin L showed a preference for larger aromatic residues in S2. In contrast, cathepsin S preferred phenylalanine to valine in S2, but bound less tightly to the V10W cystatin variant. The latter variant proved to be valuable for discriminating between cathepsin L and cathepsin S (Ki 2.4 and 190 pM respectively). The equilibrium dissociation constant of the complex between cathepsin L and variant L9W/W106G showed little difference in affinity from that of the cathepsin L complex with the singly substituted W106G variant. In contrast, the L9W/W106G variant displayed increased specificity for cathepsin S with a Ki of 10 pM. Our results clearly indicate differences in the specificity of interaction between the N-terminal region of cystatin C and cathepsins B, H, L and S, and that, although cystatin C has evolved to be a good inhibitor of all of the mammalian cysteine proteinases, more specific inhibitors of the individual enzymes can be engineered.

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Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes

Cited by 84 publications

References 30 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

Cystatin F Is a Glycosylated Human Low Molecular Weight Cysteine Proteinase Inhibitor

Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases

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