Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors 1 1Edited by A. R. Fersht

Legendre, Daniel; Laraki, Nezha; Gräslund, Torbjörn; Bjornvad, M.E.; Bouchet, Michèle; Nygren, Per‐Åke; Borchert, Torben V.; Fastrez, Jacques

doi:10.1006/jmbi.1999.3437

Cited by 37 publications

(19 citation statements)

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“…The phosphonate diester group mimics the substrate carbonyl, and the phosphorous atom is sufficiently electrophilic to permit covalent binding to the active site of serine proteases (18,19). Phosphonate diesters have recently been applied to isolate mutants of a non-Ab serine protease (subtilisin) displayed on a phage dis- play library (32). A positive charge was placed close to the phosphonate esters utilized as probes for proteolytic Abs in the present study, because most known proteolytic antibodies cleave peptide bonds on the C-terminal side of basic residues (3,(5)(6)(7)(8)31).…”

Section: Discussionmentioning

confidence: 99%

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Phosphonate Ester Probes for Proteolytic Antibodies

Paul

Tramontano

Gololobov

et al. 2001

Journal of Biological Chemistry

103

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The reactivity of phosphonate ester probes with several available proteolytic antibody (Ab) fragments was characterized. Irreversible, active site-directed inhibition of the peptidase activity was evident. Stable phosphonate diester-Ab adducts were resolved by column chromatography and denaturing electrophoresis. Biotinylated phosphonate esters were applied for chemical capture of phage particles displaying Fv and light chain repertoires. Selected Ab fragments displayed enriched catalytic activity inhibitable by the selection reagent. Somewhat unexpectedly, a phosphonate monoester also formed stable adducts with the Abs. Improved catalytic activity of phage Abs selected by monoester binding was evident. Turnover values (k cat ) for a selected Fv construct and a light chain against their preferred model peptide substrates were 0.5 and 0.2 min ؊1, respectively, and the corresponding Michaelis-Menten constants (K m ) were 10 and 8 M. The covalent reactivity of Abs with phosphonate esters suggests their ability to recapitulate the catalytic mechanism utilized by classical serine proteases. Abs1 and Ab L chains are reported to catalyze the cleavage of VIP (1, 2), the HIV coat proteins gp41 (3) and gp120 (4), Arg-vasopressin (5), thyroglobulin (6), factor VIII (7), prothrombin (8), and various model peptidase substrates (5, 9, 10). Recent studies suggest that the peptidase activity is a heritable function encoded by a germ line variable region gene(s) (11,12). In principle, the immune system may be capable of recruiting the catalyst-encoding germ line V gene(s) to elaborate specific proteolytic Abs directed to diverse polypeptide antigens, much as noncatalytic Abs capable of high affinity binding to different antigens can be developed by somatic sequence diversification of the same germ line V genes. Introduction of single replacement mutations in Ab combining sites can result in gain of proteolytic (13) and esterase (14) activities, underscoring the potential contributions of variable region diversification in maturation of Ab catalytic activities.The presence of a serine protease-like catalytic triad in a model proteolytic Ab L chain has previously been deduced from site-directed mutagenesis studies (15). Formation of a covalent complex between the nucleophilic serine residue and the substrate (the acyl-enzyme intermediate) is an essential step en route to peptide bond cleavage by non-Ab serine proteases (16). Phosphonate diesters, like the classical inhibitor DFP, can bind the active site of non-Ab serine proteases and serine esterases covalently (17)(18)(19). In comparison, negatively charged phosphonate monoesters have traditionally been assumed to bind esterolytic Abs (20, 21) and non-Ab serine esterases (22) via noncovalent electrostatic interactions. The aim of the present study was to characterize the reactivity of recombinant proteolytic Abs with phosphonate diesters and monoesters. Irreversible, active site-directed inhibition of catalytic activity by the phosphonate diesters was evident; stable Ab-phosphon...

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Section: Discussionmentioning

confidence: 99%

“…Covalent Phage Selection-A compound similar to diester II is described as permitting isolation of catalytically active subtilisin mutants from a phage library (32). Monoester III-like compounds are generally thought to bind esterolytic Abs by noncovalent electrostatic interactions (20,21).…”

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confidence: 99%

Phosphonate Ester Probes for Proteolytic Antibodies

Paul

Tramontano

Gololobov

et al. 2001

Journal of Biological Chemistry

103

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“…However, directed evolution of desired catalytic properties has proven quite a challenge. A popular strategy has been to use phage display technologies, often in combination with transition state analogues (18) or mechanism-based suicide inhibitors, for selection (30). Although successful, the enrichment conferred by these methods is generally based on binding rather than catalysis.…”

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confidence: 99%

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

Kostallas

Samuelson

2010

Appl Environ Microbiol

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We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.Proteases constitute a group of enzymes that irreversibly catalyze the cleavage of peptide bonds and represent approximately 2% of all protein-encoding genes in living organisms (39). Besides acting as virulence factors for many pathogens (16), proteases are crucial for the regulation of numerous biological processes that influence the life and death of a cell (4). These enzymes also underlie several pathological conditions, such as cancer (13) and neurodegenerative (20) and cardiovascular (8) diseases. A key issue for increasing our knowledge about such complex biological processes, and thereby hopefully also providing possibilities for new therapeutic strategies, is to deduce the proteases' substrate repertoires. Consequently, a lot of efforts around the world are dedicated to the characterization of proteases and their substrates (2, 31). In addition to their biological importance, proteases have attracted much interest in several biotechnological and industrial applications, such as removal of "fusion tags" from recombinant target proteins (38), as supplements in dishwashing and laundry detergents, or for bating of hides and skin in the leather industry (41, 44). Sometimes, however, their use is hindered due to limitations inherent to a specific protease: for example, low solubility, poor enzyme stability and specificity, or limited activity. It would therefore be of great aid to have powerful and straightforward methods available that facilitate the engineering of novel protease variants not suffering from such limitations.Traditionally, pr...

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“…Based on these structural studies, several groups have mutated the S 4 pocket in an attempt to alter the P 4 substrate specificity of subtilisin (21)(22)(23)(24)(25)(26). Wells and co-workers (27,28) found that substitutions of Asp for Tyr 104 in subtilisin BPNЈ increased cleavage of substrates containing a P 4 Arg, but the resulting mutant protease did not discriminate between Arg and Phe at this position.…”

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confidence: 99%

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

Rozan

Krysan

Rockwell

et al. 2004

Journal of Biological Chemistry

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Yeast Kex2 and human furin are subtilisin-related proprotein convertases that function in the late secretory pathway and exhibit similar though distinguishable patterns of substrate recognition. Although both enzymes prefer Arg at P 1 and basic residues at P 2 , the two differ in recognition of P 4 and P 6 residues. To probe P 4 and P 6 recognition by Kex2p, furin-like substitutions were made in the putative S 4 and S 6 subsites of Kex2. T252D and Q283E mutations were introduced to increase the preference for Arg at P 4 and P 6 , respectively. Glu 255 was replaced with Ile to limit recognition of P 4 Arg. The effects of putative S 4 and S 6 mutations were determined by examining the cleavage by purified mutant enzymes of a series of fluorogenic substrates with systematic changes in P 4 and/or P 6 . Whereas wild type Kex2 exhibited little preference for Arg at P 6 , the T252D mutant and T252D/Q283E double mutant exhibited clear interactions with P 6 Arg. Moreover, the T252D and T252D/Q283E substitutions altered the influence of the P 6 residue on P 4 recognition. We infer that cross-talk between S 4 and S 6 , not seen in furin, allows wild type and mutant forms of Kex2 to adapt their subsites for altered modes of recognition. This apparent plasticity may allow the subsites to rearrange their local environment to interact with different substrates in a productive manner. E255I-Kex2 exhibited significantly decreased recognition of P 4 Arg in a tetrapeptide substrate with Lys at P 1 , although the general pattern of selectivity for aliphatic residues at P 4 remained unchanged.The subtilisin superfamily includes a subfamily of related processing proteases, the proprotein convertases that function in the late secretory pathway of diverse eukaryotic organisms including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and mammals (1-4). Unlike the degradative subtilisins, which display a broad substrate specificity for hydrophobic residues (5), the proprotein convertases are post-translational modifying enzymes that process secretory proteins in a sequence-specific manner. In general, these proteases cleave C-terminal to clusters of basic residues, but their exact sequence specificity differs among the members of this family, even though they are Ն45% identical within their subtilisin-related domains. Similarities and differences in substrate recognition were illustrated by the enzymatic characterization of two members of this family, the S. cerevisiae protease, Kex2, and the human homologue, furin.A detailed understanding of substrate recognition by Kex2 and furin has emerged from extensive analysis of the purified secreted, soluble enzymes using model peptide substrates. Based on these studies, the consensus cleavage site for Kex2 was determined to be (Ali/Arg)-Xaa-(Lys/Arg)-Arg2 (where Ali indicates an aliphatic amino acid), with the principal determinants being a basic residue at P 2 and Arg at P 1 (6 -9).

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Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors 1 1Edited by A. R. Fersht

Cited by 37 publications

References 70 publications

Phosphonate Ester Probes for Proteolytic Antibodies

Phosphonate Ester Probes for Proteolytic Antibodies

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates

Plasticity of Extended Subsites Facilitates Divergent Substrate Recognition by Kex2 and Furin

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