Prokaryotic ubiquitin-like protein (Pup) is a functional analog of ubiquitin. Post-translationally modified pupylated proteins are selectively degraded by a proteasome-dependent proteolytic system. Deaminase of Pup (Dop) activates Pup by deaminating the C-terminal from glutamine to glutamate, and subsequently activated Pup is conjugated to target proteins by proteasome accessory factor A. Dop is also involved in the removal of Pup from pupylated proteins. Deconjugated free Pup is capable of religating to target proteins. Although the pupylation system is well studied in Mycobacterium, little is known about it in other actinomycetes. Both Rhodococcus and Mycobacterium Dop remove Pup from pupylated proteins, but in these two bacteria, no accumulation of deconjugated free Pup from Rhodococcus is observed. Analysis of a model pupylated protein revealed that Rhodococcus Pup is degraded at multiple sites by Dop. The endopeptidase activity of Dop can be detected using a fluorogenic substrate in conjunction with aminopeptidase. Moreover, the enzymatic activity of the model enzyme increases when Pup is deconjugated. These results suggest that depupylated Rhodococcus Pup is not recycled for religation with target proteins, and that Pup not only functions as a degradation signal, but also regulates the enzymatic activity of target proteins by conjugation and deconjugation to them.