کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
10537954 962885 2005 9 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Ionisations within a subtilisin-glyoxal inhibitor complex
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Ionisations within a subtilisin-glyoxal inhibitor complex
چکیده انگلیسی
Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a Ki=2.3±0.2 μM at pH 7.0 and 25 °C. Using Z-Ala-Pro-[2-13C]Phe-glyoxal we have detected a signal at 107.3 ppm by 13C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the 13C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pKa<3. This is the first observation of oxyanion formation in a reversible subtilisin-inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pKa of ∼6.5 in the free enzyme and on a pKa<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol−1. We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1749, Issue 1, 20 May 2005, Pages 33-41
نویسندگان
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