PDBsum entry 1hb0

Hydrolase

PDB id

1hb0

Contents

			Protein chain

					240 a.a. *

			Ligands

					SO4

			Metals

					_CA

			Waters ×120

* Residue conservation analysis

References listed in PDB file

Key reference

Title

X-Ray snapshots of serine protease catalysis reveal a tetrahedral intermediate.

Authors

R.C.Wilmouth, K.Edman, R.Neutze, P.A.Wright, I.J.Clifton, T.R.Schneider, C.J.Schofield, J.Hajdu.

Ref.

Nat Struct Biol, 2001, 8, 689-694. [DOI no: 10.1038/90401]

PubMed id

11473259

Abstract

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.



	Figure 3. Figure 3. Isotropic temperature factors in the acyl -enzyme complex and in the tetrahedral intermediate (b) formed between porcine pancreatic elastase and human -casomorphin-7. a, B-factors for the structure in Fig. 1a, where the PPE -BCM7 acyl -enzyme complex is stabilized at pH 5. Three N-terminal residues are disordered. b, B-factors for the structure of the tetrahedral intermediate in Fig. 1c. This structure was obtained in a freeze-quenched crystal following a 1 min long pH jump to pH 9. For data collection and refinement statistics, see Table 1.		Figure 4. Figure 4. Structural changes within the peptide binding pocket during catalysis. a, The active site cleft showing the location of the peptide substrate (pink) in the acyl -enzyme complex at pH 5. The enzyme is shown as a gray space filling model with Ser 195 (green), His 57 (purple) and Asp 102 (brown) highlighted. b, Model of the protein -peptide complex at pH 5 (pink) overlaid with the model of the tetrahedral intermediate (blue) (see Methods) . A circle highlights the active site Ser residue under the bound peptide. Both Wat 318 and hydrogen bonds between enzyme and peptide are red in the acyl -enzyme complex and blue in the tetrahedral intermediate. During product release, the loop formed by residues 217 -219 (immediately below the binding pocket) moves so as to partially fill a space previously occupied by the peptide. Arg 217 takes up a position similar to that found in the native unliganded structure (1QNJ)5.