Direct crystallographic observation of an acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of a peptidyl ester substrate: Exploiting the "glass transition" in protein dynamics. Academic Article uri icon

Overview

abstract

  • The crystal structure of the acyl complex of porcine pancreatic elastase with its peptidyl ester substrate N-acetyl-ala-ala-ala-methyl ester (Ac(Ala)3OMe) has been determined at 2.5 A resolution. The complex was stabilized by exploiting the "glass transition" in protein dynamics that occurs at around -53 degrees C (220 K). Substrate was flowed into the crystal in a cryoprotective solvent above this temperature, and then the crystal was rapidly cooled to a temperature below the transition to trap the species that formed. The use of a flow cell makes the experiment a kinetic one and means that the species prior to the rate determining transition state has a chance to accumulate. The resulting crystal structure shows an acyl-enzyme intermediate in which the leaving group is absent and the carbonyl carbon of the C-terminal alanine residue is covalently bound to the gamma oxygen of the active site serine. The ester carbonyl shows no significant distortion from planarity, with the carbonyl oxygen forming one hydrogen bond with the oxyanion hole. The tripeptide is bound in an extended antiparallel beta-sheet with main chain residues of the enzyme. The geometry and interactions of this acyl-enzyme suggest that it represents a productive intermediate. To test this hypothesis, the same crystal was then warmed above the glass transition temperature and a second data set was collected. The resulting electron density map shows no sign of the substrate, indicating hydrolysis of the intermediate followed by product release. This experiment provides direct evidence for the importance of dynamic properties in catalysis and also provides a blueprint for the stabilization of other short-lived species for direct crystallographic observation.

publication date

  • November 2, 2006

Research

keywords

  • Oligopeptides
  • Pancreatic Elastase

Identity

PubMed Central ID

  • PMC1751290

Scopus Document Identifier

  • 33750725424

Digital Object Identifier (DOI)

  • 10.1016/j.bioorg.2006.10.002

PubMed ID

  • 17083959

Additional Document Info

volume

  • 34

issue

  • 6