pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor

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Title: pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor
Authors: Howe, Nicole
Ceruso, Mariangela
Spink, Edward
Malthouse, J.Paul G.
Permanent link: http://hdl.handle.net/10197/3021
Date: Oct-2011
Abstract: The Stromelysin-1 catalytic domain83-247 (SCD) is stable for at least 16 hours at pHs 6.0-8.4. At pHs 5.0 and 9.0 there is exponential irreversible denaturation with half lives of 38 and 68 min respectively. At pHs 4.5 and 10.0 irreversible denaturation is biphasic. At 25°C, C-terminal truncation of stromelysin-1 decreases the stability of the stromelysin-1 catalytic domain at pH values > 8.4 and < 6.0. We describe the conversion of the carboxylate group of (βR)-β-[[[(1S)-1-[[[(1S)-2-Methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1'-biphenyl]-4-hexanoic acid (UK-370106-COOH) a potent inhibitor of the metalloprotease stromelysin-1 to a glyoxal group (UK-370106-CO13CHO). At pH 5.5 - 6.5 the glyoxal inhibitor is a potent inhibitor of stromelysin-1 (Ki = ~1 μM). The aldehyde carbon of the glyoxal inhibitor was enriched with carbon-13 and using Carbon-13 NMR we show that the glyoxal aldehyde carbon is fully hydrated when it is in aqueous solutions (90.4 ppm) and also when it is bound to SCD (~92.0 ppm). We conclude that the hemiacetal hydroxyl groups of the glyoxal inhibitor are not ionised when the glyoxal inhibitor is bound to SCD. The free enzyme pKa values associated with inhibitor binding were 5.9 and 6.2. The formation and breakdown of the signal at ~92 ppm due to the bound UK-370106-CO13CHO inhibitor depends on pKa values of 5.8 and 7.8 respectively. No strong hydrogen bonds are present in free SCD or in SCD-inhibitor complexes. We conclude that the inhibitor glyoxal group is not directly coordinated to the catalytic zinc atom of SCD.
Funding Details: Science Foundation Ireland
Higher Education Authority
Type of material: Journal Article
Publisher: Elsevier
Copyright (published version): 2011 Elsevier B.V.
Keywords: Metalloprotease;Glyoxal inhibitor;pH stability;pKa;Tetrahedral intermediate
Subject LCSH: Metalloproteinases--Inhibitors
Metalloproteinases--Stability
DOI: 10.1016/j.bbapap.2011.07.004
Language: en
Status of Item: Peer reviewed
Appears in Collections:Biomolecular and Biomedical Science Research Collection

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