'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes.
beta-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain gamma-lactam analogues of monocyclic beta-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrol...
| Main Authors: | , , , |
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| Format: | Journal article |
| Language: | English |
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2000
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| _version_ | 1797054262228811776 |
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| author | Wright, P Wilmouth, R Clifton, I Schofield, C |
| author_facet | Wright, P Wilmouth, R Clifton, I Schofield, C |
| author_sort | Wright, P |
| collection | OXFORD |
| description | beta-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain gamma-lactam analogues of monocyclic beta-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (beta-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its 'native' conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90 degrees from its normal position. PPE-gamma-lactam crystals were subjected to 'pH-jumps' by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the gamma-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease-inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis. |
| first_indexed | 2024-03-06T18:54:44Z |
| format | Journal article |
| id | oxford-uuid:116baca1-4e84-49d5-ba3e-1ecc06e2c780 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:54:44Z |
| publishDate | 2000 |
| record_format | dspace |
| spelling | oxford-uuid:116baca1-4e84-49d5-ba3e-1ecc06e2c7802022-03-26T10:02:18Z'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:116baca1-4e84-49d5-ba3e-1ecc06e2c780EnglishSymplectic Elements at Oxford2000Wright, PWilmouth, RClifton, ISchofield, Cbeta-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain gamma-lactam analogues of monocyclic beta-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (beta-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its 'native' conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90 degrees from its normal position. PPE-gamma-lactam crystals were subjected to 'pH-jumps' by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the gamma-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease-inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis. |
| spellingShingle | Wright, P Wilmouth, R Clifton, I Schofield, C 'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes. |
| title | 'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes. |
| title_full | 'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes. |
| title_fullStr | 'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes. |
| title_full_unstemmed | 'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes. |
| title_short | 'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes. |
| title_sort | ph jump crystallographic analyses of gamma lactam porcine pancreatic elastase complexes |
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