Tautomeric state and pKα of the phosphorylated active site histidine in the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:: sugar phosphotransferase system

The phosphorylated form of the N-terminal domain of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been investigated by one-bond and long-range H-1-N-15 correlation spectroscopy. The active site His 189 is phosphorylated at the N epsilon 2 position and has a pK(a) of 7.3, which is one pH unit higher than that of unphosphorylated His 189. Because the neutral form of unphosphorylated His 189 is in the N delta 1-H tautomer, and its N epsilon 2 atom is solvent inaccessible and accepts a hydrogen bond from the hydroxyl group of Thr 168, both protonation and phosphorylation of His 189 must be accompanied by a change in the side-chain conformation of His 189, specifically from a chi(2) angle in the g(+) conformer in the unphosphorylated state to the g(-) conformer in the phosphorylated state.