Two crystal structures of the leupeptin‐trypsin complex

Abstract

Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57.

Structural Analyses on Intermediates in Serine Protease Catalysis

“…The hemiketal oxygen atoms in this structure are very close to the location of the boronic acid oxygen atoms in the PPE complex. A similar tetrahedral conformation was observed in the complexes between chymostatin and Streptomyces griseus protease A (35), a peptidyl boronic acid inhibitor and chymotrypsin (36), and leupeptin and trypsin (37). The fact that in the TI structures described here the oxygen atoms (or oxygen and nitrogen atoms) are equidistant between the carbonyl oxygen in the acyl-enzyme intermediate gives a new view of the conformation of the tetrahedral intermediate.…”

Structural Basis of the Zinc Inhibition of Human Tissue Kallikrein 5

“…Through these hydrogen bonds, Gln192 can contribute to a better hydrolysis of bound peptide substrates. 38 Gln192 is found in many trypsin-like serine proteinases including trypsin and tryptase, and crystal structures of both proteinases complexed to leupeptin 38,39 show backbone interactions with Gln192 similar to those just described for hK5.…”

“…However, the electron density around the P1-Arg3i-Ser195 adduct unambiguously indicates that the hemiacetal group now preferentially adopts an R configuration, with the oxygen pointing out of the oxyanion hole to form a weak hydrogen bond with the N ε2 atom of the His57 imidazole ring. A similar R configuration of the hemiacetal group and out-of-oxyanion hole position of the oxygen has recently been observed in the trypsin-leupeptin complex, 39 while both configurations had been found at about equal populations in the Streptomyces griseus proteinase A complex with chymostatin. 45 In the hK5-leupeptin structure, the two conformers/configurations seem to be of similar free energy.…”

Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

“…Structures for the leupeptin-trypsin complex have been reported previously at resolutions of 1.7-1.8 Å (15). For the purposes of the present analysis, we collected data and refined the structure to 1.14 Å resolution, using the same protocols used for the acyl-enzyme structures (Table 2; PDB ID code 2AGI).…”

“…Structures of the enzyme͞substrate Michaelis complex, analogs of the tetrahedral intermediates, and acyl-enzymes can yield great insight into the atomic motions and shifting geometric relationships along the reaction coordinate. Here, we report (i) high resolution structures of trypsin acylated by two good peptide substrates and (ii) higher resolution structures of two previously solved trypsin complexes: the stable acyl-enzyme formed with the poor substrate p-nitroguanidinobenzoate (14) and the covalent complex formed with the inhibitor leupeptin, which mimics a tetrahedral intermediate (15). Comparisons provide insight into active site adjustments involved in catalysis and clarify the activation and attack trajectory of the hydrolytic water.…”