 |
|
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.
|
|
|
Authors
|
|
T.Tamada,
T.Kinoshita,
K.Kurihara,
M.Adachi,
T.Ohhara,
K.Imai,
R.Kuroki,
T.Tada.
|
|
|
Ref.
|
|
J Am Chem Soc, 2009,
131,
11033-11040.
|
|
|
PubMed id
|
|
|
|
|
|
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.
|
|
|
|