Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) is one of the most effective low M(r) inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-beta-trypsin complex

S K Sreedharan, C Verma, L S D Caves, S M Brocklehurst, S E Gharbia, H N Shah, K Brocklehurst

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1-trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was shown to inhibit beta-trypsin by a reversible competitive mechanism; this contrasts with the widely held view that E-64 is a class-specific inhibitor of the cysteine proteinases and reports in the literature that it does not inhibit a number of other enzymes including, notably, trypsin. The K-i value (3 x 10(-5) M) determined by kinetic analysis of the hydrolysis of N-alpha-benzoyl-L-arginine 4-nitroanilide in Tris/HCl buffer, pH 7.4, at 25 degrees C, I = 0.1, catalysed by beta-trypsin is comparable with those for the inhibition of trypsin by benzamidine and 4-aminobenzamidine, which are widely regarded as the most effective low M(r) inhibitors of this enzyme, Computer modelling of the beta-trypsin-E-64 adsorptive complex, by energy minimization, molecular dynamics simulation and Poisson-Boltzmann electrostatic-potential calculations, was used to define the probable binding mode of E-64; the ligand lies parallel to the active-centre cleft, anchored principally by the dominant electrostatic interaction of the guanidinium cation at one end of the E-64 molecule with the carboxylate anion of Asp-171 (beta-trypsin numbering from Ile-1) in the S-1-subsite, and by the interaction of the carboxylate substituent on C-2 of the epoxide ring at the other end of the molecule with Lys-43; the epoxide ring of E-64 is remote from the catalytic site serine hydroxy group. The possibility that E-64 might bind to the cysteine proteinases clostripain (from Clostridium histolyticum) and alpha-gingivain (one of the extracellular enzymes from Porphyromonas gingivalis) in a manner analogous to that deduced for the beta-trypsin-E-64 complex is discussed.

Original languageEnglish
Pages (from-to)777-786
Number of pages10
JournalBiochemical journal
Publication statusPublished - 15 Jun 1996



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