A clogged gutter mechanism for protease inhibitors

Radisky, Evette S.; Koshland, Daniel E.

doi:10.1073/pnas.112332899

Cited by 143 publications

(197 citation statements)

References 46 publications

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“…From the structures of intact inhibitors bound to proteases, it has widely been thought that the rigidity of the enzyme-inhibitor complexes effectively blocks the catalytic mechanism before the formation of the tetrahedral intermediate or acyl-enzyme. An alternative, or supplementary, model suggests that the acyl-enzyme intermediate forms readily, but that the peptide bond is more rapidly reformed, so that the intact form seen in crystal structures predominates (26)(27)(28)(29). This model is supported by experiments directly demonstrating formation of an acyl-enzyme intermediate by subtilisin and chymotrypsin inhibitor II (29) and analysis of the pH dependence of inhibitor hydrolysis rates (45).…”

Section: (Pdb Id Code 2age) (E) the Tetrahedral Intermediate For Hydsupporting

confidence: 50%

“…2, for both the intact inhibitor bound to wild-type trypsin (A) and the cleaved inhibitor bound to S195A trypsin (B). As observed for other enzyme-bound Laskowski inhibitors (29), the scissile amide group of the intact inhibitor displayed planar geometry, and the carbonyl carbon was ideally positioned for attack by the O␥ atom of Ser-195 (shown in red on the surface representation of the enzyme in Fig. 2).…”

Section: Resultsmentioning

confidence: 86%

“…Because the products of hydrolysis remain physically associated, the resynthesis of these inhibitors is an intramolecular reaction and is, therefore, much more favorable than the equivalent intermolecular reaction at modest reactant concentrations. Despite the prevalence of Laskowski inhibitors (25), the mechanisms by which they resist proteolysis remain poorly understood (26)(27)(28)(29).…”

mentioning

confidence: 99%

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Structure of a serine protease poised to resynthesize a peptide bond

Zakharova

Horvath

Goldenberg

2009

Proc. Natl. Acad. Sci. U.S.A.

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The serine proteases are among the most thoroughly studied enzymes, and numerous crystal structures representing the enzymesubstrate complex and intermediates in the hydrolysis reactions have been reported. Some aspects of the catalytic mechanism remain controversial, however, especially the role of conformational changes in the reaction. We describe here a high-resolution (1.46 Å) crystal structure of a complex formed between a cleaved form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site for resynthesis of the peptide bond. This structure defines the positions of the newly generated amino and carboxyl groups following the 2 steps in the hydrolytic reaction. Comparison of this structure with those representing other intermediates in the reaction demonstrates that the residues of the catalytic triad are positioned to promote each step of both the forward and reverse reaction with remarkably little motion and with conservation of hydrogen-bonding interactions. The results also provide insights into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their target proteases.trypsin ͉ bovine pancreatic trypsin inhibitor ͉ enzyme mechanisms S erine proteases are found throughout all 3 domains of cellular life and function in a wide range of physiological processes, including digestion, protein maturation and turnover, hemostasis, and immune responses (1). Approximately 0.6% of human protein-encoding genes are predicted to specify serine proteases, and this family is even more prevalent in other organisms, notably the arthropods (2, 3). A large body of biochemical and structural data have established a 2-step mechanism for hydrolysis of peptide bonds by this class of proteases (4), as shown in Scheme 1.The first step of the reaction is a nucleophilic attack by the catalytic serine residue (Ser-195 in trypsin and other members of the chymotrypsin, or S1, family) on the carbonyl carbon atom of the residue labeled P1, generating a covalent acyl-enzyme intermediate and a new peptide amino terminus, on the P1Ј residue. A second nucleophilic attack, by a water molecule, leads to hydrolysis of the acyl-enzyme, releasing the new carboxyl group and restoring the catalytic Ser residue to its initial state.

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Section: (Pdb Id Code 2age) (E) the Tetrahedral Intermediate For Hydsupporting

confidence: 50%

Section: Resultsmentioning

confidence: 86%

mentioning

confidence: 99%

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Structure of a serine protease poised to resynthesize a peptide bond

Zakharova

Horvath

Goldenberg

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…When bound, they undergo an extremely slow cleavage (Laskowskir , 1986 ;Bode and Huber , 1992 ;Radisky and Koshland , 2002 ). Canonical inhibitors are the most abundant among the serine protease inhibitors (Laskowski and Kato , 1980 ;Laskowski , 1986 ).…”

Section: Inhibition Of the S1 Family Serine Proteasesmentioning

confidence: 99%

“…They are further divided into 18 groups, based on their secondary structure (Laskowski and Qasim , 2000 ). All 18 groups share a strikingly similar backbone conformation around the scissile peptide bond, although their folds are quite different (Bode and Huber , 1992 ;Radisky and Koshland , 2002 ). Their specifi city is determined by the complementarity between the protease active-site cleft and the reactive loop of the inhibitor.…”

Section: Inhibition Of the S1 Family Serine Proteasesmentioning

confidence: 99%

β-Trefoil inhibitors – from the work of Kunitz onward

Renko

Sabotič²,

Turk³

2012

Biological Chemistry

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Protein protease inhibitors are the tools of nature in controlling proteolytic enzymes. They come in different shapes and sizes. The β -trefoil protease inhibitors that come from plants, fi rst discovered by Kunitz, were later complemented with representatives from higher fungi. They inhibit serine (families S1 and S8) and cysteine proteases (families C1 and C13) as well as other hydrolases. Their versatility is the result of the plasticity of the loops coming out of the stable β -trefoil scaffold. For this reason, they display several different mechanisms of inhibition involving different positions of the loops and their combinations. Natural diversity, as well as the initial successes in de novo protein engineering, makes the β -trefoil proteins a promising starting point for the generation of strong, specifi c, multitarget inhibitors capable of inhibiting multiple types of hydrolytic enzymes and simultaneously interacting with different protein, carbohydrate, or DNA molecules. This pool of knowledge opens up new possibilities for the exploration of their naturally occurring as well as modifi ed properties for applications in many fi elds of medicine, biotechnology, and agriculture.

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Development of a novel peptide inhibitor of subtilisin BPN′

et al. 2022

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Proteinaceous protease inhibitors can strongly and specifically inhibit cognate proteases, but their use as pharmaceuticals is limited by their size. As such, the development of effective protease peptide inhibitors would be beneficial for biochemical studies and drug discovery. In this study, we applied a phage display system to select subtilisin BPN′‐binding peptides and evaluated their inhibitory activities against subtilisin BPN′. A 12mer peptide with an intramolecular disulfide bond inhibited subtilisin BPN′ (Ki value of 13.0 nm). Further mutational analyses of the peptide resulted in the development of a short peptide inhibitor against subtilisin BPN′ that showed high inhibitory activity and binding affinity (Ki value of 0.30 nm). This activity was found to be derived from the conformational rigidity caused by the intramolecular disulfide bond and the small residue at the P1′ site and from the interaction of the P4 and P6′ residues with subtilisin BPN′.

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A clogged gutter mechanism for protease inhibitors

Cited by 143 publications

References 46 publications

Structure of a serine protease poised to resynthesize a peptide bond

Structure of a serine protease poised to resynthesize a peptide bond

β-Trefoil inhibitors – from the work of Kunitz onward

Development of a novel peptide inhibitor of subtilisin BPN′

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