How Escherichia coli purine nucleoside phosphorylase active site mutants help in understanding catalytic mechanism (CROSBI ID 543758)
Prilog sa skupa u zborniku | sažetak izlaganja sa skupa
Podaci o odgovornosti
Mikleušević, Goran ; Leščić Ašler, Ivana ; Salopek-Sondi, Branka ; Narczyk, Marta ; Luić, Marija ; Bzowska, Agnieszka
engleski
How Escherichia coli purine nucleoside phosphorylase active site mutants help in understanding catalytic mechanism
Purine nucleoside phosphorylase (PNP) is the key enzyme of purine salvage. The enzyme catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine nucleosides and some analogues: beta-purine nucleoside + orthophosphate ↔ purine base + alpha-D-pentose-1-phosphate There are two main classes of PNPs: "low-molecular" and "high-molecular" mass PNPs. High-molecular mass PNPs are hexameric enzymes occuring in various microorganisms. Especially interesting bacterial PNP is that from Escherichia coli which shown to be a promising cadidate for tumor directed gene therapy. Kinetic data for E. coli PNP revealed a complicated kinetic behaviour of this enzyme. Based on the crystal structure of a ternary complex of E. coli PNP with formycin A derivatives and phosphate or sulphate ions a putative mechanism of catalytic activity is proposed. In order to check the validity of proposed mechanism, different PNP active site mutants are prepared: D204A, D204N, R24A, R217A and a D204A/R217A double mutant. PNP mutants were overexpressed in E. coli BL21(DE3) cells and mutants were isolated and purified to ~ 99 % homogeneity. Specific activity towards inosine as a substrate of the enzyme shows 0.3 and 5.6 % of the original activity of a wild type for PNP mutants D204A and D204N, respectively. Specific activity of other mutants, as well as kinetic measurements are under way.
E. coli PNP; active site mutants
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Podaci o prilogu
67-67.
2008.
objavljeno
Podaci o matičnoj publikaciji
4th Croatian Congress of Microbiology with International Participation Book of Abstracts
Vujaklija, Dušica ; Pigac, Jasenka ; Hađina, Suzana ; Kosalec, Ivan
Zagreb: Hrvatsko mikrobiološko društvo
978-953-96567-7-3
Podaci o skupu
4th Croatian Congress of Microbiology with international participation
poster
24.09.2008-27.09.2008
Zadar, hrvatska