ABSTRACT
It is demonstrated here that in Escherichia coli, the phosphorylated form of the glucose-specific phosphocarrier protein IIAGlc of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIAGlc has no effect on the basal activity of adenylyl cyclase. To elucidate the specific role of IIAGlcphosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIAGlc were mutated by site-directed mutagenesis to glutamine and glutamate. Wild-type IIAGlc and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr∼P) and were equally potent activators of adenylyl cyclase. Neither the H90Q nor the H90E mutant of IIAGlc was phosphorylated by HPr∼P, and both failed to activate adenylyl cyclase. Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr∼P, yet the H75E mutant of IIAGlc was a partial activator of adenylyl cyclase. The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase. This suggests that the H75E mutant was transiently phosphorylated by HPr∼P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket. These results are discussed in the context of the proximity of H75 and H90 in the IIAGlc structure and the disposition of the negative charge in the modeled glutamate mutants.
Effect of some D-amino acids on the steady-state level of glutamine synthetase in Escherichia coli.
