UMP kinase from Streptococcus pneumoniae: evidence for co-operative ATP binding and allosteric regulation.
UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl2 or MgCl2, the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (K(m)=0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S0.5=9.4+/-0.7 mM and n=1.9+/-0.1 in the presence of MgCl2). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient. The kcat (175+/-13 s(-1) in the presence of MgCl2) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP.
Fassy
Florence
F
Aventis Pharma, 13 quai Jules Guesde, 94403 Vitry sur Seine Cedex, France. florence.fassy@aventis.com
Krebs
Odile
O
Lowinski
Maryse
M
Ferrari
Paul
P
Winter
Jacques
J
Collard-Dutilleul
Véronique
V
Salahbey Hocini
Khadidja
K
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site; D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002384:Catalysis / Q000187:drug effects; D002412:Cations / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011489:Protein Denaturation; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D014542:Uridine Monophosphate / Q000378:metabolism
D002412:Cations; D008024:Ligands; D015636:Magnesium Chloride; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014542:Uridine Monophosphate; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase