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A neutron laue diffraction study of endothiapepsin: Implications for the aspartic proteinase mechanism

A neutron laue diffraction study of endothiapepsin: Implications for the aspartic proteinase mechanism
A neutron laue diffraction study of endothiapepsin: Implications for the aspartic proteinase mechanism
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 Å to an R-factor of 23.5% and an Rfree 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 -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.
structural biology, enzyme mechanism, catalysis, neutron diffraction
0006-2960
13149-13157
Coates, L.
2be36c11-8cb2-4518-a001-953051aa6a23
Erskine, P.T.
c77b60c5-b80c-4e6a-a103-bf57ecfcbcf6
Wood, S.P.
430faabf-7f5c-4cf6-9bcc-5955f5e09566
Myles, D.A.A.
dee29ff3-3c05-4c17-8c4e-c418079f9b0f
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0
Coates, L.
2be36c11-8cb2-4518-a001-953051aa6a23
Erskine, P.T.
c77b60c5-b80c-4e6a-a103-bf57ecfcbcf6
Wood, S.P.
430faabf-7f5c-4cf6-9bcc-5955f5e09566
Myles, D.A.A.
dee29ff3-3c05-4c17-8c4e-c418079f9b0f
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0

Coates, L., Erskine, P.T., Wood, S.P., Myles, D.A.A. and Cooper, J.B. (2001) A neutron laue diffraction study of endothiapepsin: Implications for the aspartic proteinase mechanism. Biochemistry, 40 (44), 13149-13157. (doi:10.1021/bi010626h).

Record type: Article

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 Å to an R-factor of 23.5% and an Rfree 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 -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.

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Submitted date: 28 March 2001
Published date: 1 November 2001
Keywords: structural biology, enzyme mechanism, catalysis, neutron diffraction
Organisations: Biological Sciences

Identifiers

Local EPrints ID: 43770
URI: http://eprints.soton.ac.uk/id/eprint/43770
ISSN: 0006-2960
PURE UUID: 57dba965-433a-4fbc-bccd-7157b46b7080

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Date deposited: 01 Feb 2007
Last modified: 08 Jan 2022 15:58

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Contributors

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

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