Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein.
The uridylyltransferase/uridylyl-removing enzyme (UTase/UR) of Escherichia coli plays an important role in the regulation of nitrogen assimilation by controlling the uridylylation state of the PII signal transduction protein (PII) in response to intracellular signals. The reversible uridylylation of PII indirectly controls the activity of PII receptors that regulate transcription from nitrogen-regulated promoters and the activity of glutamine synthetase. Here, we present a detailed analysis of the uridylyltransferase and uridylyl-removing activities and their regulation by the small molecule effectors ATP, 2-ketoglutarate, and glutamine. Several important features of enzyme mechanism and regulation were elucidated. Mg2+ appeared to be the physiologically relevant metal ion cofactor for both transferase and uridylyl-removing activities. The transferase reaction proceeded by an ordered bi-bi kinetic mechanism, with PII binding before UTP and pyrophosphate (PPi) released before PII-UMP. The uridylyl-removing reaction proceeded with rapid equilibrium binding of substrate and random release of products. Both reactions were activated by ATP and 2-ketoglutarate, which did so by binding only to PII and PII-UMP. The binding of these effectors to PII and PII-UMP was characterized. Glutamine inhibited the transferase reaction by inhibiting the chemistry step, while glutamine provided nonessential mixed-type activation of the uridylyl-removing activity, lowering the apparent Km and increasing kcat. Our data were consistent with the hypothesis that all effects of glutamine are due to the binding of central complexes at a single glutamine site. By comparing the effects of the activators with their reported in vivo concentrations, we conclude that in intact cells the uridylylation state of PII is regulated mainly by the glutamine concentration and is largely independent of the 2-ketoglutarate concentration. Our kinetic data were consistent with the hypothesis that both transferase and uridylyl-removal reactions occurred at a single active center on the enzyme.
Jiang
P
P
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.
Peliska
J A
JA
Ninfa
A J
AJ
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001667:Binding, Competitive; D003570:Cytidine Triphosphate / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D005973:Glutamine / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D013379:Substrate Specificity; D014544:Uridine Triphosphate / Q000378:metabolism
D001426:Bacterial Proteins; D051922:PII Nitrogen Regulatory Proteins; D011742:Pyrimidine Nucleotides; D005973:Glutamine; D008345:Manganese; C404143:PIID regulatory protein, Bacteria; D003570:Cytidine Triphosphate; D009713:Nucleotidyltransferases; D009612:Nicotinamide-Nucleotide Adenylyltransferase; C022515:regulatory protein uridylyltransferase; D005974:Glutamate-Ammonia Ligase; D014544:Uridine Triphosphate