Now showing 1 - 2 of 2
  • Publication
    pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor
    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.
      499Scopus© Citations 2
  • Publication
    The importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin
    Two new inhibitors have synthesized where the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced by a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H respectively. Using these inhibitors we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases inhibitor binding 3-4 fold while a glyoxal group increases binding by 500-2000 fold. We show that at pH 7.2 the effective molarity of the catalytic hydroxyl group of the active site serine is 41,000-229,000 and 101,000 to159,000 for α-chymotrypsin and subtilisin Carlsberg respectively. It is estimated that oxyanion stabilisation and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pKa of the active site histidine enabling it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed.
      500Scopus© Citations 15