title: Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-beta-trypsin complex.
abstract: 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 K1, 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 Mr inhibitors of this enzyme. Computer modelling of the beta-trypsin-E64 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 S1-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 phyromonas gingivalis) in a manner analogous to that deduced for the beta-trypsin-E-64 complex is discussed.
authors.affiliation: Department of Biochemistry, Queen Mary and Westfield College, University of London, U.K.
authors.forename: S K
authors.initials: SK
authors.lastname: Sreedharan
authors.forename: C
authors.initials: C
authors.lastname: Verma
authors.forename: L S
authors.initials: LS
authors.lastname: Caves
authors.forename: S M
authors.initials: SM
authors.lastname: Brocklehurst
authors.forename: S E
authors.initials: SE
authors.lastname: Gharbia
authors.forename: H N
authors.initials: HN
authors.lastname: Shah
authors.forename: K
authors.initials: K
authors.lastname: Brocklehurst
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D002417:Cattle; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D012984:Software; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: D014361:Trypsin Inhibitors; D014357:Trypsin; D007930:Leucine; C024974:E 64