INHIBITION OF BOVINE BETA-TRYPSIN, HUMAN ALPHA-THROMBIN AND PORCINE PANCREATIC BETA-KALLIKREIN-B BY 4',6-DIAMIDINO-2-PHENYLINDOLE, 6-AMIDINOINDOLE AND BENZAMIDINE - A COMPARATIVE THERMODYNAMIC AND X-RAY STRUCTURAL STUDY

The inhibitory effect of 4',6-diamidino-2-phenylindole (DAPI) and 6-am idinoindole on the catalytic properties of bovine beta-trypsin (trypsi n), human alpha-thrombin (thrombin) and porcine pancreatic beta-kallik rein-B (kallikrein) was investigated (between pH 3.0 and 7.0, I = 0.1 M; T = 30.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine p roteinases. Next, the X-ray crystal structure of the trypsin:DAPI comp lex was solved at 1.9 Angstrom resolution (R = 0.161). Over the whole pH range explored, values of the association inhibition constant (K-i) for DAPI and 6-amidinoindole binding to trypsin, thrombin and kallikr ein are higher than those found for benzamidine association, suggestin g a binding mode of DAPI to the enzyme primary specificity pocket-base d on the indole moiety of the inhibitor. On lowering the pH from 5.5 t o 3.0, the decrease in affinity for DAPI, 6-amidinoindole and benzamid ine binding to trypsin, thrombin and kallikrein reflects the acidic pK shift of the Asp189 invariant residue, present at the bottom of the p rimary 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 comp lex, however, does not allow a unique interpretation of the inhibitor binding mode. The present data were analysed in parallel with those re ported for related serine (pro)enzyme/inhibitor systems.