EFFECT OF PHOSPHORYLATION ON HYDROGEN-BONDING INTERACTIONS OF THE ACTIVE-SITE HISTIDINE OF THE PHOSPHOCARRIER PROTEIN HPR OF THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM DETERMINED BY N-15 NMR-SPECTROSCOPY

The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His 15. The hydrogen-bonding state of this histidine was examined with 15N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with 15N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the Nδ1 and Nє2 positions or at only the Nδ1 position. 15N NMR spectra of two synthesized model compounds, phosphoimidazole and phosphomethylimidazole, were also recorded. We show that, prior to phosphorylation, the protona ted His 15 Nє2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated Nδ1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His 15 Nδ1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed. © 1990, American Chemical Society. All rights reserved.