title: 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.
abstract: 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.
authors: Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195, Japan.
authors: Taro
authors: T
authors: Tamada
authors: Takayoshi
authors: T
authors: Kinoshita
authors: Kazuo
authors: K
authors: Kurihara
authors: Motoyasu
authors: M
authors: Adachi
authors: Takashi
authors: T
authors: Ohhara
authors: Keisuke
authors: K
authors: Imai
authors: Ryota
authors: R
authors: Kuroki
authors: Toshiji
authors: T
authors: Tada
mesh_terms: D000818:Animals; D000838:Anions; D002384:Catalysis; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D033363:Neutron Diffraction; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011487:Protein Conformation; D013552:Swine; D014961:X-Ray Diffraction
chemical_list: D000838:Anions; D010196:Pancreatic Elastase