Robustness of the Escherichia coli Signal-Transducing UTase/UR-PII Covalent Modification Cycle to Variation in the PII Concentration Requires Very Strong Inhibition of the UTase Activity of UTase/UR by Glutamine

摘要

Uridylyltransferase/Uridylyl-Removing Enzyme (UTase/UR) catalyzes uridylylation of PII and deuridylylation of PII-UMP, with both activities regulated by glutamine. In a reconstituted UTase/UR-PII cycle containing wild-type UTase/UR, the steady-state modification of PII varied from nearly complete modification to nearly complete demodification as glutamine was varied, whether PII was saturating or unsaturating. But when a his-tagged version of UTase/UR was used, robustness to variations in PII concentration was lost, and the range of PII modification states in response to glutamine became smaller as PII concentration increased. The presence of the his-tag on UTase/UR did not alter PII substrate inhibition of the UT activity and had little effect on the level of the UT activity, but resulted in a slight defect in the UR activity. Importantly, at high PII concentration, glutamine inhibition of the UT activity was incomplete. We hypothesized that PII binding to the UR active site in the HD domain was responsible for PII substrate inhibition of the UT activity and, in the his-tagged enzyme, also diminished glutamine inhibition of the UT activity. Consistent with this, three different UTase/UR proteins with HD domain alterations lacked substrate inhibition of the UT activity by PII; in one case the HD alteration eliminated glutamine regulation of the UT activity, while for the other two proteins, alterations of the HD domain partially compensated for the effect of the his-tag in restoring glutamine regulation of the UT activity. We conclude that very strong inhibition of the UT activity was required for the UTase/UR-PII cycle to display robustness to the PII concentration, that in the wild-type enzyme PII brings about substrate inhibition of the UT activity by binding to the HD domain of the enzyme, and that addition of an N-terminal His-tag resulted in an altered enzyme with subtle changes in the interactions between domains such that PII binding to the HD domain interfered with glutamine regulation of the UT domain.