Naslov
Homooligomerization is needed for stability: a molecular modelling and solution study of E. coli purine nucleoside phosphorylase

Autori
Bertoša, Branimir ; Mikleušević, Goran ; Wielgus-Kutrowska, Beata ; Narczyk, Marta ; Hajnić, Matea ; Leščić Ašler, Ivana ; Tomić, Sanja ; Luić, Marija ; Bzowska, Agnieszka

Izvornik
The FEBS journal (1742-464X) 281 (2014), 7; 1860-1871

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
homooligomeric proteins; molecular modelling; obligate (obligatory) oligomer; analytical ultracentrifugation; CD spectra; purine nucleoside phosphorylase

Sažetak
Although many enzymes are homooligomers composed of tightly bound subunits, it is often the case that smaller assemblies of such subunits, or even individual monomers, seem to have all the structural features necessary to independently conduct catalysis. In this study, we investigated the reasons justifying the necessity for the hexameric form of E. coli purine nucleoside phosphorylase - a homohexamer composed of three linked dimers - since it appears that the dimer is the smallest unit capable of catalyzing the reaction, according to the currently accepted mechanism. Molecular modelling was employed to probe mutations at the dimer-dimer interface that would result in a dimeric enzyme form. In this way, both in silico and in vitro, the hexamer was successfully transformed into dimers. However, modelling and solution studies show that, when isolated, dimers cannot maintain the appropriate three-dimensional structure, including the geometry of the active site and the position of the catalytically important amino acids. Analytical ultracentrifugation proves that E. coli purine nucleoside phosphorylase dimeric mutants tend to dissociate into monomers with dissociation constants of 20-80 M. Consistently, the catalytic activity of these mutants is negligible, at least 6 orders of magnitude smaller than for the wild type enzyme. We conclude that the hexameric architecture of E. coli purine nucleoside phosphorylase is necessary to provide stabilization of the proper three-dimensional structure of the dimeric assembly, and therefore this enzyme is the obligate (obligatory) hexamer.

Izvorni jezik
Engleski

Znanstvena područja
Fizika, Kemija, Biologija

POVEZANOST RADA