We utilized the non-pathogenic M. smegmatis strain as a model and systematically investigated the dynamic proteome changes in response to the overexpressing of MsKat/MtCobB in mycobacteria. After that, the quantitative mass spectrometric method was utilized and showed that the AMP-dependent synthetase, Citrate synthase, ATP-dependent specificity component of the Clp protease, and ATP-dependent DNA/RNA helicases were identified to be the substrates of MsKat.