Open AccessUMP kinase from the Gram‐positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity
Author(s) -
Gagyi Cristina,
Bucurenci Nadia,
Sîrbu Ovidiu,
Labesse Gilles,
Ionescu Mihaela,
Ofiteru Augustin,
Assairi Liliane,
Landais Stéphanie,
Danchin Antoine,
Bârzu Octavian,
Gilles AnneMarie
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03702.x
Subject(s) - bacillus subtilis , biochemistry , gtp' , kinase , nucleoside diphosphate kinase , enzyme , escherichia coli , biology , chemistry , microbiology and biotechnology , bacteria , gene , genetics
The gene encoding Bacillus subtilis UMP kinase ( pyrH / smbA ) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units·mg −1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3′‐anthraniloyl‐2′‐deoxyguanosine‐5′‐triphosphate (Ant‐dGTP) can increase catalysis significantly. Binding of Ant‐dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant‐dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5‐fluoro‐UMP (5F‐UMP) and 6‐aza‐UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N ‐acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.