Journal
BIOORGANIC & MEDICINAL CHEMISTRY
Volume 20, Issue 22, Pages 6758-6769Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bmc.2012.08.045
Keywords
One-third-of-the-sites; Enzyme catalysis; Enzyme inhibitors; Homooligomeric proteins; ITC; Negative cooperativity; Transition state; Spectroscopic methods
Funding
- Polish Ministry of Science and Higher Education [N301 003 31/0042, N N301 044939, BW-1724/BF]
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Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [ Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon. (C) 2012 Elsevier Ltd. All rights reserved.
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