ENZYMOLOGICAL CHARACTERIZATION OF THE SIGNAL-TRANSDUCING URIDYLYLTRANSFERASE URIDYLYL-REMOVING ENZYME (EC-2.7.7.59) OF ESCHERICHIA-COLI ANDITS INTERACTION WITH THE PII PROTEIN/

The uridylyltransferase/uridylyl-removing enzyme (UTase/UR) of Escheri chia coli plays an important role in the regulation of nitrogen assimi lation by controlling the uridylylation state of the PII signal transd uction protein (PII) in response to intracellular signals. The reversi ble uridylylation of PII indirectly controls the activity of PII recep tors that regulate transcription from nitrogen-regulated promoters and the activity of glutamine synthetase. Here, we present a detailed ana lysis of the uridylyltransferase and uridylyl-removing activities and their regulation by the small molecule effecters ATP, 2-ketoglutarate, and glutamine. Several important features of enzyme mechanism and reg ulation were elucidated. Mg2+ appeared to be the physiologically relev ant metal ion cofactor for both transferase and uridylyl-removing acti vities. The transferase reaction proceeded by an ordered bi-bi kinetic mechanism, with PII binding before UTP and pyrophosphate (PPI) releas ed 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 effecters to PII and PII-UMP was characterized. Glutamine inhibited the transferase re action by inhibiting the chemistry step, while glutamine provided none ssential mixed-type activation of the uridylyl-removing activity, lowe ring the apparent K-m and increasing k(cat). Our data were consistent with the hypothesis that all effects of glutamine are due to the bindi ng of central complexes at a single glutamine site. By comparing the e ffects of the activators with their reported in vivo concentrations, w e conclude that in intact cells the uridylylation state of PII is regu lated mainly by the glutamine concentration and is largely independent of the 2-ketoglutarate concentration. Our kinetic data were consisten t with the hypothesis that both transferase and uridylyl-removal react ions occurred at a single active center on the enzyme.