PDBsum entry 2agi

Hydrolase/hydrolase inhibitor

PDB id: 2agi

Contents

Protein chain

223 a.a.

Ligands

ACE-LEU-LEU-AR7

SO4 ×2

Metals

_CA

Waters ×371

References listed in PDB file

Key reference

Title

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

Authors

E.S.Radisky, J.M.Lee, C.J.Lu, D.E.Koshland.

Ref.

Proc Natl Acad Sci U S A, 2006, 103, 6835-6840. [DOI no: 10.1073/pnas.0601910103]

PubMed id

16636277

Abstract

Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction.

Figure 1.
Fig. 1. Stick diagrams overlaid with 2F[o]-F[c] density maps contoured at 1 (gray mesh), showing covalent attachment of substrate ligands to trypsin. Oxygens are colored red and nitrogens are colored blue for all structures; carbons are color-coded differently for the enzyme and ligand residues of each structure, as detailed below. (A) For AAPR-trypsin, enzyme carbons are green, and substrate carbons are yellow. (B) For AAPK-trypsin, enzyme carbons are light blue, and substrate carbons are tan. Both conformations of Ser-195 are shown. (C) For the leupeptin-trypsin hemiacetal, enzyme carbons are purple, and substrate carbons are orange. Both hemiacetal conformations are shown. (D) For guanidinobenzoyl-trypsin, enzyme carbons are teal, and substrate carbons are brown. The guanidinobenzoyl moiety is substantially rotated in the active site compared with the other substrate ligands. Consequently, positioning of the figure to clearly display substrate density removes the His-57 side chain from the viewing slab.

Figure 2.
Fig. 2. Structural views and superpositions focusing on enzyme and substrate residues and water molecules involved in reaction. (A) Stereoviews comparing AAPR-trypsin (enzyme carbons, green; substrate carbons and water molecule, yellow) and leupeptin-trypsin (enzyme carbons, purple; substrate carbons, orange) with a representative trypsin/inhibitor Michaelis complex (enzyme carbons, gray; inhibitor carbons, brick red) allow reconstruction of a probable reaction coordinate for the acylation reaction. (B) Stereoviews comparing AAPR-trypsin, AAPK-trypsin (enzyme carbons, light blue; substrate carbons and water molecule, tan), and leupeptin-trypsin structures allow reconstruction of a probable reaction coordinate for water attack in the deacylation reaction. (C) Stereoviews comparing AAPR-trypsin and guanidinobenzoyl-trypsin (enzyme carbons, teal; inhibitor carbons, brown) reveal differences in attack trajectory possibly responsible for the great differences in reactivity between these substrates. (D and E) AAPR-trypsin (D) and AAPK-trypsin (E), stick diagrams overlaid with 2F[o]-F[c] density maps contoured at 1 (gray mesh) and F[o]-F[c] maps scaled at 3 (green mesh) reveal dual conformations of His-57, as well as well defined density for water S-25. (F) Stereoviews of superimposed acyl-enzyme structures of different serine proteases, demonstrating a conserved position for the proposed hydrolytic water. Carbons and the attacking water molecule are colored differently for each structure: suc-AAPR-trypsin (green), suc-AAPK-trypsin (yellow), Ac-NPI-elastase [aqua; PDB ID code 1GVK (28)], and -chymotrypsin [magenta; PDB ID code 2GCT (29)].