Combined high-resolution neutron and X-ray analysis of inhibited elastase confirms the active-site oxyanion hole but rules against a low-barrier hydrogen bond

J Am Chem Soc. 2009 Aug 12;131(31):11033-40. doi: 10.1021/ja9028846.


To help resolve long-standing questions regarding the catalytic activity of the serine proteases, the structure of porcine pancreatic elastase has been analyzed by high-resolution neutron and X-ray crystallography. To mimic the tetrahedral transition intermediate, a peptidic inhibitor was used. A single large crystal was used to collect room-temperature neutron data to 1.65 A resolution and X-ray data to 1.20 A resolution. Another crystal provided a low-temperature X-ray data set to 0.94 A resolution. The neutron data are to higher resolution than previously reported for a serine protease and the X-ray data are comparable with other studies. The neutron and X-ray data show that the hydrogen bond between His57 and Asp102 (chymotrypsin numbering) is 2.60 A in length and that the hydrogen-bonding hydrogen is 0.80-0.96 A from the histidine nitrogen. This is not consistent with a low-barrier hydrogen which is predicted to have the hydrogen midway between the donor and acceptor atom. The observed interaction between His57 and Asp102 is essentially a short but conventional hydrogen bond, sometimes described as a short ionic hydrogen bond. The neutron analysis also shows that the oxygen of the oxopropyl group of the inhibitor is present as an oxygen anion rather than a hydroxyl group, supporting the role of the "oxyanion hole" in stabilizing the tetrahedral intermediate in catalysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anions
  • Catalysis
  • Catalytic Domain
  • Hydrogen Bonding
  • Neutron Diffraction*
  • Pancreatic Elastase / antagonists & inhibitors
  • Pancreatic Elastase / chemistry*
  • Protein Conformation
  • Swine
  • X-Ray Diffraction*


  • Anions
  • Pancreatic Elastase