title: pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor.
abstract: The stromelysin-1 catalytic domain(83-247) (SCD) is stable for at least 16 h 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-CO(13)CHO). At pH 5.5-6.5 the glyoxal inhibitor is a potent inhibitor of stromelysin-1 (K(i)=~1μM). 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 (90.4ppm) and also when it is bound to SCD (~92.0ppm). 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 pK(a) values associated with inhibitor binding were 5.9 and 6.2. The formation and breakdown of the signal at ~92ppm due to the bound UK-370106-CO(13)CHO inhibitor depends on pK(a) 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.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland.
authors.forename: Nicole
authors.initials: N
authors.lastname: Howe
authors.forename: Mariangela
authors.initials: M
authors.lastname: Ceruso
authors.forename: Edward
authors.initials: E
authors.lastname: Spink
authors.forename: J Paul G
authors.initials: JP
authors.lastname: Malthouse
mesh_terms: D002208:Caproates / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D020134:Catalytic Domain / Q000502:physiology; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D006037:Glyoxal / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019278:Matrix Metalloproteinase 3 / Q000737:chemistry* / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D008954:Models, Biological; D019906:Nuclear Magnetic Resonance, Biomolecular; D011083:Polycyclic Compounds; D011485:Protein Binding; D011522:Protons; D013696:Temperature; D014633:Valine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: C476600:3-(((2,2-dimethyl-1-(((2-methoxy-1-phenylethyl)amino)carbonyl)propyl)amino)carbonyl)-6-(3-methyl-4-phenylphenyl)hexanoic acid; D002208:Caproates; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D011083:Polycyclic Compounds; D011522:Protons; D006037:Glyoxal; D019278:Matrix Metalloproteinase 3; D014633:Valine