 |
PDBsum entry 1gkt
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Hydrolase/hydrolase inhibitor
|
PDB id
|
|
|
|
1gkt
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
A neutron laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism.
|
|
|
Authors
|
|
L.Coates,
P.T.Erskine,
S.P.Wood,
D.A.Myles,
J.B.Cooper.
|
|
|
Ref.
|
|
Biochemistry, 2001,
40,
13149-13157.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Current proposals for the catalytic mechanism of aspartic proteinases are
largely based on X-ray structures of bound oligopeptide inhibitors possessing
nonhydrolyzable analogues of the scissile peptide bond. However, the positions
of protons on the catalytic aspartates and the ligand in these complexes have
not been determined with certainty. Thus, our objective was to locate crucial
protons at the active site of an inhibitor complex since this will have major
implications for a detailed understanding of the mechanism of action. We have
demonstrated that high-resolution neutron diffraction data can be collected from
crystals of the fungal aspartic proteinase endothiapepsin bound to a transition
state analogue (H261). The neutron structure of the complex has been refined at
a resolution of 2.1 A to an R-factor of 23.5% and an R(free) of 27.4%. This work
represents the largest protein structure studied to date by neutron
crystallography at high resolution. The neutron data demonstrate that 49% of the
main chain nitrogens have exchanged their hydrogen atoms with D2O in the mother
liquor. The majority of residues resisting exchange are buried within core
beta-sheet regions of the molecule. The neutron maps confirm that the protein
has a number of buried ionized carboxylate groups which are likely to give the
molecule a net negative charge even at very low pH, thereby accounting for its
low pI. The functional groups at the catalytic center have clearly undergone H-D
exchange despite being buried by the inhibitor occupying the active site cleft.
Most importantly, the data provide convincing evidence that Asp 215 is
protonated and that Asp 32 is the negatively charged residue in the transition
state complex. This has an important bearing on mechanistic proposals for this
class of proteinase.
|
|
|
|
|
|
|
