PDBsum entry 1lw6

Hydrolase

PDB id: 1lw6

Contents

Protein chains

281 a.a. *

63 a.a. *

Ligands

SO4 ×4

Metals

_CA

Waters ×505

* Residue conservation analysis

References listed in PDB file

Key reference

Title

A clogged gutter mechanism for protease inhibitors.

Authors

E.S.Radisky, D.E.Koshland.

Ref.

Proc Natl Acad Sci U S A, 2002, 99, 10316-10321. [DOI no: 10.1073/pnas.112332899]

PubMed id

12142461

Abstract

A classical peptide inhibitor of serine proteases that is hydrolyzed approximately 10(7) times more slowly than a good substrate is shown to form an acyl-enzyme intermediate rapidly. Despite this quick first step, further reaction is slowed dramatically because of tight and oriented binding of the cleaved peptide, preventing acyl-enzyme hydrolysis and favoring the reverse reaction. Moreover, this mechanism appears to be common to a large class of tight-binding serine protease inhibitors that mimic good substrates. The arrest of enzymatic reaction at the intermediate stage allowed us to determine that the consensus nucleophilic attack angle is close to 90 degrees in the reactive Michaelis complexes.

Figure 3.
Fig 3. (A) Ribbon diagram of subtilisin/CI2 complex structure. Subtilisin is shown in red, the N-terminal section of CI2 is shown in dark blue, and the C-terminal section of CI2 is shown in light blue. The reactive site peptide bond is at the junction of the dark and light blue segments. (B) Closer view of reactive site loop. CI2 side chains (labeled in white) and hydrogen bonds (yellow dotted lines) proposed to stabilize the positioning of the light blue (leaving group) side of the loop after acyl-enzyme formation (see text) are shown in detail. The serine, histidine, and aspartate of the subtilisin catalytic triad are also shown (labeled in yellow).

Figure 4.
Fig 4. Nucleophilic attack trajectories for protease/inhibitor complexes. (A) The geometric parameters describing the nucleophilic attack trajectory are diagrammatically defined. [y] is the angle defined by the enzyme serine -oxygen, the inhibitor carbonyl carbon, and the inhibitor carbonyl oxygen. [x] is the angle between (i) the plane defined by the enzyme serine -oxygen, the inhibitor carbonyl carbon, and the inhibitor carbonyl oxygen, and (ii) the plane defined by the peptide bond. O--C represents the distance between the enzyme serine -oxygen and the inhibitor carbonyl carbon. (B) Plot of [y] vs. [x]. Blue triangles represent the structures of 78 protease/inhibitor complexes, the orange circle represents the subtilisin/CI2 complex, and the red square represents the thrombin/fibrinogen analog structure (16). The peptide bond diagrammed in the background is for illustrative purposes. (C and D) Two views of the superposition of 79 protease/inhibitor complexes, including subtilisin/CI2. Superpositioning was based on the -carbon and carbonyl oxygen of the P[1] residue, and the amide nitrogen of the P residue, which overlay closely for all structures. The red spheres represent the relative positions of the enzyme serine -oxygen for each structure. The outlying structure apparent in B, C, and D is that of an ecotin mutant complexed with trypsin.