pH-dependent binding modes observed in trypsin crystals: Lessons for structure-based drug design

ChemBioChem

Volumn 3, Issue 2-3, 2002, Pages 246-249

  Stubbs, Milton T ^a   Reyda, Sabine ^a   Dullweber, Frank ^a   Möller, Maren ^a   Klebe, Gerhard ^a   Dorsch, Dieter ^b   Mederski, Werner W K R ^b   Wurziger, Hanns ^b  

^a PHILIPPS UNIVERSITY MARBURG   (Germany)

^b MERCK KGAA   (Germany)

Author keywords

Crystal structure; Drug research; Factor Xa; Induced fit; Inhibitors

Indexed keywords

ARTICLE; CHEMICAL BINDING; CRYSTAL STRUCTURE; CRYSTALLIZATION; DISSOCIATION CONSTANT; DRUG RESEARCH; HYDROGEN BOND; MOLECULAR INTERACTION; PH; PRIORITY JOURNAL; STRUCTURE ANALYSIS;

EID: 0036523422     PISSN: 14394227     EISSN: None     Source Type: Journal    
DOI: 10.1002/1439-7633(20020301)3:2/3<246::aid-cbic246>3.0.co;2-%23     Document Type: Article

References (21)

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9. i values for bovine β-trypsin were determined photometrically under the following conditions: 0.05 M ttris(hydroxymethyl)aminomethane/HCl (tris/ HCl; pH 8.0) 0.154 M NaCl, and 5% ethanol, with three different concentrations of the Factor Xa substrate Pefachrome tPA (Pentapharm, Basel) at 25°C. For the standard protocols used, see: J. Stürzebecher, U. Stürzebecher, H. Vieweg, G. Wagner, J. Hauptmann, F. Markwardt, Thromb. Res. 1989, 54, 245-252.
10. d were determined by isothermal titration experiments with an MCS-ITC instrument (Microcal Inc., USA) as described in: T. Wiseman, S. Williston, J. F. Brandts, L. N. Lin, Anal. Biochem. 1989, 179, 131-137. The inhibitor (0.5 mM) was titrated into the trypsin solution (0.04 mM) in the reaction cell with a 250 μL syringe at 25°C. Experiments were carried out at pH 5.0 in 50 mM β-morpholinoethanesulfonic acid buffer solution and at pH 7.8 in 50 mM tris buffer solution, in each case with 100 mM sodium chloride and 5% ethanol. Injection volumes were 15 μL and 4 min equilibration time was allowed between each injection. Observations were corrected for the heats of dilution of the inhibitor in the buffer solution.
11. [10c] The final model for A corresponds to an R factor of 19.0% for reflections between 10 and 3.0 Å (99.9% completeness); the model for B yields an R factor of 20.3% from 10 to 2.1 Å (96.6% completeness). Coordinates, structure factors, and all relevant statistical data have been deposited at the Protein Data Bank (accession codes: 1q18 and 1q17 for the A and B forms, respectively). a) Z. Otkinowski, W. Minor, Methods Enzymol. A 1997, 276, 307-326; b) A. Brünger, X-PLOR (Version 3.1), Yale University Press, New Haven, 1992; c) T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeelgaard, Acta Crystallogr. 1991, 47, 110-119.
12. [10c] The final model for A corresponds to an R factor of 19.0% for reflections between 10 and 3.0 Å (99.9% completeness); the model for B yields an R factor of 20.3% from 10 to 2.1 Å (96.6% completeness). Coordinates, structure factors, and all relevant statistical data have been deposited at the Protein Data Bank (accession codes: 1q18 and 1q17 for the A and B forms, respectively). a) Z. Otkinowski, W. Minor, Methods Enzymol. A 1997, 276, 307-326; b) A. Brünger, X-PLOR (Version 3.1), Yale University Press, New Haven, 1992; c) T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeelgaard, Acta Crystallogr. 1991, 47, 110-119.
13. [10c] The final model for A corresponds to an R factor of 19.0% for reflections between 10 and 3.0 Å (99.9% completeness); the model for B yields an R factor of 20.3% from 10 to 2.1 Å (96.6% completeness). Coordinates, structure factors, and all relevant statistical data have been deposited at the Protein Data Bank (accession codes: 1q18 and 1q17 for the A and B forms, respectively). a) Z. Otkinowski, W. Minor, Methods Enzymol. A 1997, 276, 307-326; b) A. Brünger, X-PLOR (Version 3.1), Yale University Press, New Haven, 1992; c) T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeelgaard, Acta Crystallogr. 1991, 47, 110-119.
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15. Chymotrypsinogen numbering is used, therefore there is no residue 218 in trypsin.
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