Substrate Specificity and Chemical Mechanism for the Reaction Catalyzed by Glutamine Kinase

Campylobacter jejuni, a leading cause of gastroenteritis world wide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (L-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of L-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of L-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated (Enz-X-Pβ-Pγ) and phosphorylated (Enz-X-Pβ) intermediates. In the absence of L-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reacti...