and Discussion Framework of Cleaved BPTI Bound to Trypsin.

and Discussion Framework of Cleaved BPTI Bound to Trypsin. BPTI* complexes with resolution limits of 1 1.49 and 1.46 ? respectively. Structures of the complexes were determined by the method of molecular replacement using the structure of the complex of rat trypsin and intact BPTI decided at 1.8-? resolution (35). Refinement yielded R values of 18.7% and 17.5% for complexes of trypsin with BPTI and BPTI* respectively. Refinement and data collection statistics are provided in SI Table Isochlorogenic acid B manufacture 1. The structure of the active-site regions of the enzyme and inhibitor are shown in Fig. 2 for both the intact inhibitor bound to wild-type trypsin (A) and the cleaved inhibitor bound to S195A trypsin (B). As observed for other enzyme-bound Laskowski inhibitors (29) the scissile amide group of the intact inhibitor displayed planar geometry and the carbonyl carbon was ideally positioned for attack by the Oγ atom of Ser-195 (shown in red on the surface representation of the enzyme in Fig. 2). The electron density map calculated for the complex made up of cleaved BPTI showed the newly generated amino and carboxyl groups in well-defined positions in the enzyme active site. Hydrolysis of the peptide bond was accommodated by a small change in the conformation of Lys-15 and a displacement Rabbit Polyclonal to SLC27A5. of ≈1 ? in the position of the nitrogen atom of Ala-16. The electron density maps in the region of Cys-38 of the cleaved inhibitor indicated the presence of 2 side-chain conformations one nearly identical to that seen in the intact inhibitor and the other differing by rotation of the χ1 dihedral angle by ?100° thereby changing the chirality of the Cys-14-Cys-38 disulfide bond. The occupancy of the altered conformation was estimated to be ≈20%. This disulfide isomerization has been detected at a very low level (≈5%) by NMR spectroscopy in free intact BPTI (36 37 and in a crystal structure of a BPTI mutant with 3 amino acid replacements in the trypsin-binding loop (38 39 The alternate isomer is usually accommodated within the constraints of the complex with essentially no perturbation of Cys-14 or the backbone of either Isochlorogenic acid B manufacture Cys residue. Beyond the active-site region the structures of the enzyme and inhibitor were essentially identical in the 2 2 complexes. High-Resolution Reconstruction of the Serine-Protease Mechanism. Together with previously decided crystal structures of enzyme-inhibitor complexes and acyl-enzyme intermediates the structure of BPTI* bound to trypsin contributes to a detailed structural description of the steps making up the entire serine protease mechanism. Fig. 2C shows a superposition of the catalytic residues of 4 structures: the BPTI-trypsin complex (carbon atoms colored green) the BPTI*-S195A trypsin complex (orange carbon atoms) bovine trypsin bound to a tetrahedral transition-state analog (purple carbon atoms) (16) and an acyl-enzyme intermediate formed by bovine trypsin and a peptide-nitroanilide substrate (gray carbon atoms) (13). The close superposition of the catalytic residues in the 4 structures suggests that the reaction proceeds with minimal structural changes in the active site. A reconstruction of the peptide hydrolysis reaction is usually illustrated in Fig. 3 using the 4 superimposed structures described above. For clarity only the side chains of Ser-195 and His-57 are shown along with the scissile peptide unit or the boronate transition-state analog. To illustrate the geometry of potential hydrogen bonds involving the Nε2 atom of His-57 a hydrogen atom bound to this site was added to each model by using standard geometry. As noted previously the structure of the BPTI-trypsin complex shown in Fig. 3A displays all of the features expected of a successful enzyme-substrate complicated using the Ser-195 Oγ atom located to strike the carbonyl carbon from the substrate (indicated with the arrow) as well as the Nε2 atom of His-57 located to activate the Ser air by agreeing to its.