- Bacteria belonging to the phyla Actinobacteria and Nitrospira possess proteasome cores homologous to the eukaryotic 20S proteasome particle. In these bacteria, the cytoplasmic signal for proteasomal degradation is a small protein termed Pup (prokaryotic ubiquitin‐like protein). PafA, the only known Pup ligase, conjugates Pup to lysine side chains of target proteins. In contrast to the eukaryotic ubiquitin‐proteasome system, where poly‐ubiquitin chains are the principal tags for proteasomal degradation, mono‐Pup moieties are almost exclusively observed in vivo and are sufficient as degradation tags. Although Pup presents lysines, raising the possibility of poly‐Pup chain assembly, these do not predominate. At present, the factors promoting the distinct predominance of mono‐ over poly‐pupylation remain poorly understood. To address this issue, we conducted a detailed biochemical analysis characterizing the pupylation of model proteins in vitro. We found that Pup can indeed serve as a pupylation target for PafA either in its free form or when already conjugated to proteins, thus allowing for the formation of poly‐Pup chains. However, our results indicate that pupylation of an already pupylated protein is unlikely to occur due to low affinity of PafA for such species. This alone prevents predominance of poly‐ over mono‐pupylation in vitro. This effect is likely to be magnified in vivo by the combination of PafA kinetics with the high abundance of non‐pupylated proteins. Overall, this work provides a kinetic explanation for the prevalence of mono‐ rather than poly‐pupylation in vivo, and sheds light on PafA substrate specificity.