2 CRYSTAL-STRUCTURES OF THE LEUPEPTIN-TRYPSIN COMPLEX

Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The fir st structure was determined at 1.7 Angstrom resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dim ensions of a = b = 55.62 Angstrom, c = 110.51 Angstrom. The second str ucture was determined at a resolution of 1.8 Angstrom with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell di mensions of a = 63.69 Angstrom, b = 69.37 Angstrom, c = 63.01 Angstrom . The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 Angstrom. The leupep tin backbone forms four hydrogen bonds with trypsin and a fifth hydrog en bond interaction is mediated by a water molecule. The aldehyde carb onyl of leupeptin forms a covalent bond of 1.42 Angstrom length with s ide-chain oxygen of Ser-195 in the active site. The reaction of trypsi n with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxy anion hole and forming a hydrogen bond with His-57.