title: Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate.
abstract: In Mycobacterium tuberculosis, the enzyme PafA is responsible for the activation and conjugation of the proteasome-targeting molecule Pup to protein substrates. As the proteasomal pathway has been shown to be vital to the persistence of M. tuberculosis, understanding the reaction mechanism of PafA is critical to the design of antituberculous agents. In this study, we have developed novel techniques to study the activity of PafA and have characterized fundamental features of the reaction mechanism. We show that PafA catalyzes a two-step reaction mechanism proceeding through a γ-glutamyl phosphate-mixed anhydride intermediate that is formed on the C-terminal glutamate of Pup before transfer of Pup to the substrate acceptor lysine. SDS-PAGE analysis of formation of the phosphorylated intermediate revealed that the rate of Pup activation matched the maximal steady-state rate of product formation in the overall reaction and suggested that Pup activation was rate-limiting when all substrates were present at saturating concentrations. Following activation, both ADP and the phosphorylated intermediate remained associated with the enzyme awaiting nucleophilic attack by a lysine residue of the target protein. The PafA reaction mechanism appeared to be noticeably biased toward the stable activation of Pup in the absence of additional substrate and required very low concentrations of ATP and Pup relative to other carboxylate-amine/ammonia ligase family members. The bona fide nucleophilic substrate PanB showed a 3 orders of magnitude stronger affinity than free lysine, promoting Pup conjugation to occur close to the rate limit of activation with physiologically relevant concentrations of substrate.
authors.affiliation: Institute of Molecular Biology & Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Ethan
authors.initials: E
authors.lastname: Guth
authors.forename: Michael
authors.initials: M
authors.lastname: Thommen
authors.forename: Eilika
authors.initials: E
authors.lastname: Weber-Ban
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation / Q000502:physiology; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C533288:Pup protein, Mycobacterium tuberculosis; D014452:Ubiquitins; D000244:Adenosine Diphosphate; D044767:Ubiquitin-Protein Ligases