Inhibition of bovine β-trypsin, human α-thrombin and porcine pancreatic β-kallikrein-B by 4′,6-diamidino-2-phenylindole, 6-amidinoindole and benzamidine: a comparative thermodynamic and X-ray structural study

Elena Casale, Charles Collyer, Paolo Ascenzi, Gianni Balliano, Paola Milla, Franca Viola, Mauro Fasano, Enea Menegatti, Martino Bolognesi

Research output: Contribution to journalArticlepeer-review

Abstract

The inhibitory effect of 4′,6-diamidino-2-phenylindole (DAPI) and 6-amidinoindole on the catalytic properties of bovine β-trypsin (trypsin), human α-thrombin (thrombin) and porcine pancreatic β-kallikrein-B (kallikrein) was investigated (between pH 3.0 and 7.0, I = 0.1 M; T = 30.0 ± 0.5°C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Next, the X-ray crystal structure of the trypsin: DAPI complex was solved at 1.9 Å resolution (R = 0.161). Over the whole pH range explored, values of the association inhibition constant (Ki) for DAPI and 6-amidinoindole binding to trypsin, thrombin and kallikrein are higher than those found for benzamidine association, suggesting a binding mode of DAPI to the enzyme primary specificity pocket-based on the indole moiety of the inhibitor. On lowering the pH from 5.5 to 3.0, the decrease in affinity for DAPI, 6-amidinoindole and benzamidine binding to trypsin, thrombin and kallikrein reflects the acidic pK shift of the Asp189 invariant residue, present at the bottom of the primary specificity subsite of the serine proteinases considered, from 4.5, in the free enzyme, to 3.7, in the proteinase:inhibitor complexes. Inspection of the refined crystal structure of the trypsin: DAPI complex, however, does not allow a unique interpretation of the inhibitor binding mode. The present data were analysed in parallel with those reported for related serine (pro)enzyme/inhibitor systems.

Original languageEnglish
Pages (from-to)75-81
Number of pages7
JournalBiophysical Chemistry
Volume54
Issue number1
DOIs
Publication statusPublished - 1995

Keywords

  • 4′,6-Diamidino-2-phenylindole
  • Bovine β-trypsin
  • Human α-thrombin
  • Porcine pancreatic β-kallikrein-B
  • Thermodynamics of serine proteinase:inhibitor complex
  • X-ray crystal structure

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Organic Chemistry
  • Physical and Theoretical Chemistry

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