Naslov
Escherichia coli purine nucleoside phosphorylase validation of catalytic mechanism

Autori
Narczyk, Marta ; Wielgus-Kutrowska, Beata ; Mikleušević, Goran ; Leščić Ašler, Ivana ; Luić, Marija ; Bzowska, Agnieszka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Tenth International Summer School on Biophysics: Supramolecular Structure and Function, Book of Abstracts / Greta Pifat-Mrzljak and Ksenija Zahratka - Zagreb : Institut Ruđer Bošković, 2009, 135-135

ISBN
978-953-6690-81-7

Skup
TENTH INTERNATIONAL SUMMER SCHOOL ON BIOPHYSICS: SUPRAMOLECULAR STRUCTURE AND FUNCTION

Mjesto i datum
Rovinj, Hrvatska, 19.09.2009. - 01.10.2009

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
purine nucleoside phosphorylase; active site mutants; kinetics; catalysis

Sažetak
Purine nucleoside phosphorylase (PNP) is a key enzyme in metabolic pathways of purines in cells. The enzyme catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine nucleosides and some analogues. PNP from Escherichia coli, in contrast to the human enzyme, is a hexamer and has broad substrate specificity, and therefore is especially interesting, because it has shown to be a good candidate in gene therapy against solid tumours*. The crystal structure of a ternary complex of E. coli PNP with formycin A derivatives and phosphate or sulphate ions shows that the hexameric molecule is in fact the trimer of dimers. In two monomers forming the dimer the active sites are found in two - open and closed - conformations, these conformations consecutively follow each other**. Studies in solution show that binding of phosphate is described by two dissociation constants Kd, one for strong binding with Kd = 29.4 μ M and one for much weaker binding with Kd = 1.12 mM, and relative populations of about 50% each**. The stoichiometry of binding of formycin A derivatives suggest, that it happens also in the case of the nucleoside substrate**. Based on these results, a putative mechanism of the catalytic reaction is proposed: the main role is attributed to Asp204 that protonates N(7) of the purine base, and to Arg217 which cooperates with Asp204. The residue Arg24 from the neighbour subunit in the dimer is responsible for binding of phosphate ion**.In this work, the results of kinetic, fluorimetric and calorimetric studies of interaction of E. coli PNP mutants with substrates and inhibitors will be presented. To check the validity of proposed catalytic mechanism, PNP active site mutants are prepared: Asp204Ala, Asp204Asn, Arg24Ala, Arg217Ala and Asp204Ala/Arg217Ala double mutant. The preliminary kinetic studies suggest that all residues (Asp204, Arg24, Arg217) are important for catalytic activity, since their mutation into Ala lowers the activity vs natural purine nucleoside substrates (Ino, Guo, Ado) by at least 100-fold, while activity vs 7-methylated substrate (m7Guo) is even 3-fold higher than of the wild type enzyme. This proves the proposed N(7)protonation mechanism**. Residues Asp204 and Arg217 in fact seem to cooperate with each other in the process of protonation of the purine base since the double mutant has similar activity as Asp204Ala mutant, i.e. one mutation has the same effect as two mutations. The fluorimetric and calorimetric studies of interaction of PNP with Formycins A and B (close structural analogues of natural substrates adenosine and inosine, with noncleavable C-C glicosidic bond) let us measure the binding constants, stoichiometry, the enthalpy and entropy changes. Estimating the different energetic contributions to the binding will explain the themodynamic mechanism of reaction. Preliminary calorimetric studies of PNP wild type binding formycin A confirm that binding of inhibitor is described by two dissociation constants, first is in the micromolar range, the second is definitely weaker and the binding in the second case seems to be entropy driven.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

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