Proteasome accessory factor A (PafA) transferase activity makes sense in the light of its homology with glutamine synthetase

Nir Hecht, Ofir Regev, Daniel Dovrat, Amir Aharoni, Eyal Gur

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


The Pup-proteasome system (PPS) is a prokaryotic tagging and degradation system analogous in function to the ubiquitin-proteasome system (UPS). Like ubiquitin, Pup is conjugated to proteins, tagging them for proteasomal degradation. However, in the PPS, a single Pup-ligase, PafA, conjugates Pup to a wide variety of proteins. PafA couples ATP hydrolysis to formation of an isopeptide bond between Pup and a protein lysine via a mechanism similar to that used by glutamine synthetase (GS) to generate glutamine from ammonia and glutamate. GS can also transfer the glutamyl moiety from glutamine to a hydroxyl amine in an ATP-independent manner. Recently, the ability of PafA to transfer Pup from one protein to another was demonstrated. Here, we report that such PafA activity mechanistically resembles the transferase activity of GS. Both PafA and GS transferase activities are ATP-independent and proceed in two catalytic steps. In the first step catalyzed by PafA, an inorganic phosphate is used by the enzyme to depupylate a Pup donor, while forming an acyl phosphate Pup intermediate. The second step consists of Pup conjugation to the new protein, alongside the release of an inorganic phosphate. Detailed experimental analysis, combined with kinetic modeling of PafA transferase activity, allowed us to correctly predict the kinetics and magnitude of Pup transfer between two targets, and analyze the effects of their affinity to PafA on the efficiency of transfer. By deciphering the mechanism of the PafA transferase reaction in kinetic detail, this work provides in-depth mechanistic understanding of PafA, a key PPS enzyme.

Original languageEnglish
Pages (from-to)668-681
Number of pages14
JournalJournal of Molecular Biology
Issue number5
StatePublished - 2 Mar 2018


  • Glutamine synthetase
  • PafA
  • Pupylation
  • Transferase
  • Transpupylation

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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