Naslov
Validation of the catalytic mechanism of E. coli purine nucleoside phosphorylase

Autori
Luić, Marija ; Štefanić, Zoran ; Mikleušević, Goran ; Narczyk, Marta ; Wielgus-Kutrowska, Beata ; Bzowska, Agnieszka

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

Izvornik
The 5th Central European Conference-Chemistry towards Biology, Book of Abstracts / Abramić, Marija ; Maksić, Zvonimir ; Salopek-Sondi, Branka ; Tomić, Sanja ; Vianello, Robert. - Zagreb : Institut Ruđer Bošković, 2010, 40-40

ISBN
13 978-953-6690-83-1

Skup
The 5th Central European Conference-Chemistry towards Biology

Mjesto i datum
Primošten, Hrvatska, 08.09.2010. - 11.09.2010

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Nije recenziran

Ključne riječi
purine nucleoside phosphorylase; active site mutants; mechanism of catalysis; X-ray diffraction; conformational change

Sažetak
Purine nucleoside phosphorylase (PNP) is the key enzyme in the purine salvage pathway. It catalyses the reversible phosphorolytic cleavage of the glycosydic bond of purine nucleosides and some analogues. Biologically active form of the Escherichia coli purine nucleoside phosphorylase (PNP) is a homohexamer, whose structure could be described as a trimer of dimers. In order to validate a catalytic mechanism proposed for this enzyme, five active site mutants: Arg24Ala, Asp204Ala, Asp204Asn, Arg217Ala and Asp204Ala/Arg217Ala were prepared. All mutated residues are very important for the catalytic activity, since their change into alanine reduces activity of the enzyme by at least 100-fold. Activity of the mutants vs natural substrates adenosine, inosine and guanosine as well as 7-methylguanosine confirms that catalysis involves protonation of the purine base at the position N7 by the side chain of the Asp204. Kinetic studies as well as the crystal structures of wild type and Arg24Ala mutant in complexes with phosphate are carried out and their results will be presented. These results provide insight into the structure and catalytic mechanism of E. coli PNP. Since E. coli PNP has shown to be a promising candidate for tumour-directed gene therapy, this may help in design mutants useful for medical use.

Izvorni jezik
Engleski

Znanstvena područja
Fizika, Kemija, Biologija

POVEZANOST RADA