Catalyzes the covalent attachment of the prokaryotic ubiquitin-like protein modifier Pup to the proteasomal substrate proteins, thereby targeting them for proteasomal degradation. This tagging system is termed pupylation. The ligation reaction involves the side-chain carboxylate of the C-terminal glutamate of Pup and the side-chain amino group of a substrate lysine. PafA is required to confer resistance against the lethal effects of reactive nitrogen intermediates (RNI), antimicrobial molecules produced by activated macrophages and other cell types. ATP + [prokaryotic ubiquitin-like protein]-L-glutamate + [protein]-L-lysine = ADP + phosphate + N(6)-([prokaryotic ubiquitin-like protein]-gamma-L-glutamyl)-[protein]-L-lysine. Protein degradation; proteasomal Pup-dependent pathway. Protein modification; protein pupylation. Interacts with the prokaryotic ubiquitin-like protein Pup. Cells lacking this gene have no detectable pupylated proteins and substrate proteins accumulate. These cells also become hypersensitive to reactive nitrogen intermediates (RNI) and fail to grow in both wild-type and nitric oxide synthase 2 deficient macrophages. Moreover, they display increased resistance to hydrogen peroxide. The reaction mechanism probably proceeds via the activation of Pup by phosphorylation of its C-terminal glutamate, which is then subject to nucleophilic attack by the substrate lysine, resulting in an isopeptide bond and the release of phosphate as a good leaving group. Belongs to the Pup ligase/Pup deamidase family. Pup-conjugating enzyme subfamily.