PDBsum entry 1gkt

Hydrolase/hydrolase inhibitor

PDB id: 1gkt

Contents

Protein chain

329 a.a. *

Ligands

BOC-HIS-PRO-PHE-HIS-LOV-ILE-HIS

DOD ×41

Waters ×214

* Residue conservation analysis

PDB id:

1gkt

Name:

Hydrolase/hydrolase inhibitor

Title:

Neutron laue diffraction structure of endothiapepsin complexed with transition state analogue inhibitor h261

Structure:

Endothiapepsin. Chain: a. Inhibitor, h261. Chain: b. Engineered: yes

Source:

Cryphonectria parasitica. Chestnut blight fungus. Organism_taxid: 5116. Synthetic: yes. Synthetic construct. Organism_taxid: 32630

Biol. unit:

Hetero-Dimer (from PDB file)

Authors:

L.Coates,P.T.Erskine,S.P.Wood,D.A.A.Myles,J.B.Cooper

Key ref:

L.Coates et al. (2001). A neutron Laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism. Biochemistry, 40, 13149-13157. PubMed id: 11683623 DOI: 10.1021/bi010626h

Date:

20-Aug-01 Release date: 20-Nov-01

Protein chain

P11838  (CARP_CRYPA) -  Endothiapepsin from Cryphonectria parasitica

Seq: 419 a.a.

Struc: 329 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.23.22 - endothiapepsin.
• Reaction: Hydrolysis of proteins with broad specificity similar to that of pepsin A, preferring hydrophobic residues at P1 and P1', but does not cleave 14-Ala-|-Leu-15 in the B chain of insulin or Z-Glu-Tyr. Clots milk.

DOI no: 10.1021/bi010626h

Biochemistry 40:13149-13157 (2001)

PubMed id: 11683623

A neutron Laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism.

L.Coates, P.T.Erskine, S.P.Wood, D.A.Myles, J.B.Cooper.

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.