Trimeric purine nucleoside phosphorylase: Exploring postulated one-third-of-the-sites binding in the transition state

• Published in: Bioorganic & medicinal chemistry Vol. 20; no. 22; pp. 6758 - 6769
• Main Authors: Wielgus-Kutrowska, Beata, Breer, Katarzyna, Hashimoto, Mariko, Hikishima, Sadao, Yokomatsu, Tsutomu, Narczyk, Marta, Dyzma, Alicja, Girstun, Agnieszka, Staroń, Krzysztof, Bzowska, Agnieszka
• Format: Journal Article
• Language: English
• Published: OXFORD Elsevier Ltd 15.11.2012; Elsevier
• Subjects: Animals; Binding Sites; Biochemistry & Molecular Biology; Biological and medical sciences; Calorimetry; calves; Catalytic Domain; Cattle; Chemistry; Chemistry, Medicinal; Chemistry, Organic; Cooperativity; Enzyme catalysis; Enzyme inhibitors; Escherichia coli; Fluorometry; Homooligomeric proteins; hypoxanthine; Hypoxanthine - chemistry; Hypoxanthine - metabolism; ITC; Life Sciences & Biomedicine; Ligands; Medical sciences; Negative cooperativity; nucleosides; One-third-of-the-sites; Pharmacology & Pharmacy; Pharmacology. Drug treatments; phosphorylase; Physical Sciences; protein subunits; Purine Nucleosides - chemistry; Purine Nucleosides - metabolism; Purine-Nucleoside Phosphorylase - chemistry; Purine-Nucleoside Phosphorylase - genetics; Purine-Nucleoside Phosphorylase - metabolism; Pyrimidinones - chemistry; Pyrimidinones - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Science & Technology; Spectroscopic methods; stoichiometry; Thermodynamics; Transition state; (S)-PMP-DAP; 9-deaza-Gua; Negative cooperativity; DFPP-DG; Kieq; Ino; One-third-of-the-sites; m7Guo; Homooligomeric proteins; DFTHPEP-H; Hx; Kiapp; Gua; PNP; Enzyme inhibitors; Enzyme catalysis; DFPP-G; Transition state; aza-DFPP-DG; Spectroscopic methods; Kd; ITC; ImmH; (S)-PMP-Ada; CRYSTAL-STRUCTURE; DIFLUOROMETHYLENE PHOSPHONIC ACID; 9-DEAZAGUANINE; 7-METHYLGUANOSINE; PURIFICATION; INTERMEDIATE; MULTISUBSTRATE ANALOG INHIBITORS; DERIVATIVES; CALF SPLEEN; Trimer; Molecular cooperativity; Enzyme; Transferases; Glycosyltransferases; Enzyme inhibitor; Binding site; Purine-nucleoside phosphorylase; Protein; Isothermal titration calorimetry; Pentosyltransferases
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/j.bmc.2012.08.045

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.