Asymmetry in the autophosphorylation of the two-component regulatory system transmitter protein nitrogen regulator II of Escherichia coli.
Autophosphorylation of the homodimeric two-component system transmitter protein nitrogen regulator II (NRII; also NtrB) of Escherichia coli is the first step in the activation of nitrogen-regulated (Ntr) gene transcription. We show that the autophosphorylation of NRII was asymmetric, with phosphorylation of the first and second subunits of the dimer displaying different equilibria (under our experimental conditions K(1) approximately 0. 345, K(2) approximately 0.0044). Phosphorylation of both subunits of NRII was rapid, but the very rapid reversal of the phosphorylation of the second subunit was responsible for the equilibrium position of the reaction. Complete phosphorylation of NRII was only observed under conditions where ADP, a product of the autophosphorylation reaction, was removed by an enzymatic system. Purified, doubly phosphorylated NRII (NRII approximately P(2)) was stable in the absence of nucleotides at 0 degrees C but was dephosphorylated to the hemiphosphorylated form at 37 degrees C. In the presence of a low concentration of ADP, half of the phosphoryl groups from NRII approximately P(2) were rapidly dephosphorylated, while the remaining phosphoryl groups were slowly dephosphorylated. Experiments with heterodimers containing wild-type and mutant, nonphosphorylatable subunits suggested that the asymmetry of NRII autophosphorylation was not preexisting but resulted from the autophosphorylation of one subunit.
Jiang
P
P
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Peliska
J A
JA
Ninfa
A J
AJ
D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
D011494:Protein Kinases; C055699:protein kinase-phosphatase NTRB; D010749:Phosphoprotein Phosphatases