An examination of the inhibitory mechanism of serpins by analysing the interaction of trypsin and chymotrypsin with α2-antiplasmin

Enghild, Jan J.; Valnickova, Zuzana; Thøgersen, Ida B.; Pizzo, Salvatore V.; Salvesen, Guy S.

doi:10.1042/bj2910933

Cited by 48 publications

(53 citation statements)

References 22 publications

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“…Partitioning between the inhibitor and substrate pathways is seen for the interaction of serpins with some chymotrypsin family members (Patston et al, 1991;Gettins et al, 1992;Cooperman et al, 1993;Enghild et al, 1993;Schechter et al, 1993). However, it is not usually as extreme as shown here with the subtilisins unless specific mutations are made in the serpin that cause it to favor the substrate pathway (see Hood et al, 1994, for example).…”

Section: Discussionmentioning

confidence: 99%

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Interaction of subtilisins with serpins

et al. 1996

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Serpins are well-characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that a'proteinase inhibitor inhibits subtilisin Curlsberg and proteinase K, and alantichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0-45 "C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.

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Section: Discussionmentioning

confidence: 99%

“…Occasionally, cleavage outside the P I inhibitory site, but within the RSL, causes partitioning (Schechter et al, 1989;Enghild et al, 1993). For the interaction of subtilisins with a l P I , both appear to occur and there are two components of the partitioning.…”

Section: Discussionmentioning

confidence: 99%

Interaction of subtilisins with serpins

et al. 1996

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“…Heparin modulation of serpin reactivity by induced conformational change was interpreted to support a preequilibrium model [17], as was the temperature dependence of the cleavage of the reactive site loop of a2AP by trypsin and chymotrypsin [54]. Recent studies showed identical immunoreactivity to antibodies, raised against cleaved ATIII, of either partially denatured, protease-111 121…”

Section: Referencesmentioning

confidence: 99%

Structural aspects of serpin inhibition

et al. 1994

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The essential roles of proteins of the serpin family in many physiological processes, along with new discoveries of their unique folding properties, have attracted intense interest in recent years. Many serpins display unusual mobile behavior attributed to rearrangements of a-helical or /J-sheet domains, whereby large scale transitions accompany a variety of functions, including inactivation. This unusual behavior was first recognized with the X-ray structure of modified al-proteinase inhibitor. Subsequent experiments, including new X-ray structures, have revealed a surprising variety of conformations which are functionally important but only partially understood. We review here experimental evidence for conformations relevant to the serpin inhibitory mechanism.

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“…Non-covalent Bond of SCCA1 and Papain-Although an SDS-resistant complex between SCCA1 and papain was not detected, it would be possible that the covalent bond between these two proteins is unstable to SDS, like the complex between chymotrypsin and ␣ 2 -antiplasmin (21). To retain the native association between SCCA1 and papain, we employed a gelfiltration system.…”

Section: Expression and Purification Of Functional Scca1mentioning

confidence: 99%

Inhibitory Mechanism of a Cross-class Serpin, the Squamous Cell Carcinoma Antigen 1

Masumoto

Sakata

Arima

et al. 2003

Journal of Biological Chemistry

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The squamous cell carcinoma antigen (SCCA) 1 and its homologous molecule, SCCA2, belong to the ovalbuminserpin family. Although SCCA2 inhibits serine proteinases such as cathepsin G and mast cell chymase, SCCA1 targets cysteine proteinases such as cathepsin S, K, L, and papain. SCCA1 is therefore called a cross-class serpin. The inhibitory mechanism of the standard serpins is well characterized; those use a suicide substrate-like inhibitory mechanism during which an acyl-enzyme intermediate by a covalent bond is formed, and this complex is stable against hydrolysis. However, the inhibitory mechanism of cross-class serpins remains unresolved. In this article, we analyzed the inhibitory mechanism of SCCA1 on a cysteine proteinase, papain. SCCA1 interacted with papain at its reactive site loop, which was then cleaved, as the standard serpins. However, gel-filtration analyses showed that SCCA1 did not form a covalent complex with papain, in contrast to other serpins. Interaction with SCCA1 severely impaired the proteinase activity of papain, probably by inducing conformational change. The decreased, but still existing, proteinase activity of papain was completely inhibited by SCCA1 according to the suicide substrate-like inhibitory mechanism; however, papain recovered its proteinase activity with the compromised level, when all of intact SCCA1 was cleaved. These results suggest that the inhibitory mechanism of SCCA1 is unique among the serpin superfamily in that SCCA1 performs its inhibitory activity in two ways, contributing the suicide substrate-like mechanism without formation of a covalent complex and causing irreversible impairment of the catalytic activity of a proteinase.The serpins (serine proteinase inhibitors) are a superfamily of proteinase inhibitors characterized by a conserved structure and employing a suicide substrate-like inhibitory mechanism (1, 2). The structure of the serpins consists of three ␤ sheets (A-C), nine ␣ helices (A-I), and the reactive site loop (RSL) 1 composed of ϳ17 amino residues (1). The inhibitory mechanism of the serpin is well characterized (2). The exposed RSL of the serpin is recognized by the proteinase, and an initial noncovalent Michaelis encounter complex is formed. Then, in the inhibitory pathway, a "bait" peptide bond (P1-P1Ј) that mimics the normal substrate of the proteinase is attacked by the active serine residue of the proteinase, subsequently forming an acylenzyme intermediate linked by an oxy-ester bond. In the cleaved form, the P side of the RSL inserts into the body of the protein, which dramatically changes the conformations of the serpin and the proteinase, making it impossible for the ester bond to hydrolyze (3). In the non-inhibitory or substrate pathways, the serpin is cleaved by the proteinase just as the substrate of the proteinase after the Michaelis encounter complex is formed. It has been revealed that the serpins are involved in various kinds of biological functions: fibrinolysis, coagulation, inflammation, tumor cell invasion, cellular differentiat...

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An examination of the inhibitory mechanism of serpins by analysing the interaction of trypsin and chymotrypsin with α2-antiplasmin

Cited by 48 publications

References 22 publications

Interaction of subtilisins with serpins

Interaction of subtilisins with serpins

Structural aspects of serpin inhibition

Inhibitory Mechanism of a Cross-class Serpin, the Squamous Cell Carcinoma Antigen 1

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