UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity.
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.
Gagyi
Cristina
C
Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, Paris, France.
Bucurenci
Nadia
N
Sîrbu
Ovidiu
O
Labesse
Gilles
G
Ionescu
Mihaela
M
Ofiteru
Augustin
A
Assairi
Liliane
L
Landais
Stéphanie
S
Danchin
Antoine
A
Bârzu
Octavian
O
Gilles
Anne-Marie
AM
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D016153:Genes, Suppressor; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; C078379:pyrH protein, E coli; D006160:Guanosine Triphosphate; D014166:Transferases; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase