Design, Chemical Synthesis and Kinetic Studies of Trypsin Chromogenic Substrates Based on the Proteinase Binding Loop of Cucurbita maxima Trypsin Inhibitor (CMTI-III)

Lesner, Adam; Brzozowski, Krzysztof; Kupryszewski, G.; Rolka, Krzysztof

doi:10.1006/bbrc.2000.2257

Cited by 18 publications

(9 citation statements)

References 19 publications

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“…toluenosulfonyl or acetyl) was present at the N-terminus, and an amide moiety was attached to the aromatic ring. The results obtained correspond with our previous studies [5,20], where acetylation of the N-terminal -amino group of trypsin chromogenic substrates was shown to increase the substrate specificity towards this enzyme. The same effect was observed for chymotrypsin and HLE.…”

Section: Resultssupporting

confidence: 91%

“…Three to five measurements were carried out for each compound (systematic error expressed as a standard deviation never exceeded 10%). All details of kinetic studies and the method of calculating kinetic parameters, Michaelis constants (K M ), catalytic constants (k cat ), and specificity constants (k cat \K M ) have been described in our previous papers [5,20].…”

Section: Methodsmentioning

confidence: 99%

“…Originally, fragments of naturally occurring protease inhibitors flanking the scissile peptide bond were used as starting structures of fluoro-and chromogenic substrates [5]. However, such an approach is restricted to a limited number of natural molecules.…”

mentioning

confidence: 99%

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Selection of New Chromogenic Substrates of Serine Proteinases Using Combinatorial Chemistry Methods

Wysocka

Kwiatkowska²,

Rzadkiewicz³

et al. 2007

CCHTS

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Chemical synthesis, physicochemical characterization and kinetic investigations of a tetrapeptide library of chromogenic substrates containing the amide of 5-amino-2-nitrobenzoic acid (Anb(5,2)-NH(2)) at their C-termini are reported. Anb(5,2)-NH(2) served as a chromophore released upon enzymatic action. The library consisting of 9567 peptides was synthesized using the portioning-mixing method and was screened against bovine a-chymotrypsin and human leukocyte elastase in solution applying an iterative approach. The selected chromogenic substrates were resynthesized and further modified at their N- and C-termini. Finally, two sequences, Z-Phe-Ala-Thr-Tyr-Anb(5,2)-NH(2) and Z-Phe-Phe-Pro-Val-Anb(5,2)-NH(2), were obtained as highly specific substrates for bovine alpha-chymotrypsin and human leukocyte elastase, respectively. The method of synthesis and selection of chromogenic substrates of serine proteinases described herein is straightforward and can be applied to design substrates for other proteases.

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Section: Resultssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

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Selection of New Chromogenic Substrates of Serine Proteinases Using Combinatorial Chemistry Methods

Wysocka

Kwiatkowska²,

Rzadkiewicz³

et al. 2007

CCHTS

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“…The hydrolysis sites were determined by high-performance liquid chromatography (HPLC)-MS and the kinetics of hydrolysis were measured as previously described [30,31]. …”

Section: Methodsmentioning

confidence: 99%

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

et al. 2013

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Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1’) with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.

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“…All details of kinetic studies and the method of calculating kinetic parameters, the Michaelis (K M ), catalytic (k cat ), and specificity constants (k cat /K M ) have been described in our previous papers [14,15]. The concentration of bovine -trypsin stock solution was determined by titration with NPGB.…”

Section: Kinetic Studiesmentioning

confidence: 99%

Designing of Substrates and Inhibitors of Bovine α-Chymotrypsin with Synthetic Phenylalanine Analogues in Position P1

Lesner¹,

Wysocka²,

Łęgowska³

et al. 2008

PPL

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The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.

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Design, Chemical Synthesis and Kinetic Studies of Trypsin Chromogenic Substrates Based on the Proteinase Binding Loop of Cucurbita maxima Trypsin Inhibitor (CMTI-III)

Cited by 18 publications

References 19 publications

Selection of New Chromogenic Substrates of Serine Proteinases Using Combinatorial Chemistry Methods

Selection of New Chromogenic Substrates of Serine Proteinases Using Combinatorial Chemistry Methods

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

Designing of Substrates and Inhibitors of Bovine α-Chymotrypsin with Synthetic Phenylalanine Analogues in Position P1

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Design, Chemical Synthesis and Kinetic Studies of Trypsin Chromogenic Substrates Based on the Proteinase Binding Loop of Cucurbita maxima Trypsin Inhibitor (CMTI-III)

Cited by 18 publications

References 19 publications

Selection of New Chromogenic Substrates of Serine Proteinases Using Combinatorial Chemistry Methods

Selection of New Chromogenic Substrates of Serine Proteinases Using Combinatorial Chemistry Methods

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

Designing of Substrates and Inhibitors of Bovine &#945;-Chymotrypsin with Synthetic Phenylalanine Analogues in Position P1

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Designing of Substrates and Inhibitors of Bovine α-Chymotrypsin with Synthetic Phenylalanine Analogues in Position P1