title: Trimeric purine nucleoside phosphorylase: exploring postulated one-third-of-the-sites binding in the transition state.
abstract: 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.
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Żwirki i Wigury 93, Warsaw, Poland.
authors.forename: Beata
authors.initials: B
authors.lastname: Wielgus-Kutrowska
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Breer
authors.forename: Mariko
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Sadao
authors.initials: S
authors.lastname: Hikishima
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Yokomatsu
authors.forename: Marta
authors.initials: M
authors.lastname: Narczyk
authors.forename: Alicja
authors.initials: A
authors.lastname: Dyzma
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Girstun
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Staroń
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Bzowska
mesh_terms: D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D020134:Catalytic Domain; D002417:Cattle; D005470:Fluorometry; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D011684:Purine Nucleosides / Q000737:chemistry / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011744:Pyrimidinones / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D008024:Ligands; D011684:Purine Nucleosides; D011744:Pyrimidinones; D011994:Recombinant Proteins; D019271:Hypoxanthine; C113101:forodesine; D011683:Purine-Nucleoside Phosphorylase