In the present DFT study, mechanisms of peptide hydrolysis by Co(III)- and Cu(II)-containing complexes of 1,4,7,10-tetraazacyclododecane (cyclen), 1-Co and 1-Cu, respectively, and 1-oxa-4,7,10-triazacyclododecane (oxacyclen), 2-Co and 2-Cu, respectively, and their analogues have been investigated. In addition, the effects of the ligand environment, pendant (an organic group containing a recognition site) and metal ion (Co(III), Cu(II), Ni(II), Zn(II), Cd(II), and Pd(II)), on the energetics of this reaction have been elucidated. The reactant of the 1-Co complex exists in the syn–anti conformation, while that of 1-Cu in the syn–syn form. For both these complexes, stepwise and concerted mechanisms were found to occur with similar barriers. The substitution of one of the nitrogen atoms in the cyclen macrocycle to create oxacyclen should occur at position 10 in the Co(III) case and at position 4 in the Cu(II) case. A comparison between the barriers using the common conformation (syn–anti) of 1-Co and 2-Co showed that both complexes hydrolyze the peptide bond with similar barriers, i.e., 39.8 kcal/mol for the former and 40.1 kcal/mol for the latter. This result is in line with the measured data that suggest that the oxacyclen complex exhibits just four times greater activity than the cyclen complex. The removal of the pendant (−C2H5) group in the Co(III)- and Cu(II)-cyclen complexes (1′-Co and 1′-Cu, respectively) reduced the barriers by 9.3 and 3.0 kcal/mol, respectively. For 1′-Co, the barrier of 30.5 kcal/mol is in agreement with the experimental value of 25.9 kcal/mol for the cleavage of myoglobin at pH 9.0 and 50 °C. The reactants of 1′-Cu, 1′-Zn, 1′-Pd, and 1′-Cd adopt the syn–syn conformation, whereas 1′-Ni and 1′-Co exist in the syn–anti geometry. The barriers for 1′-Ni (triplet spin state), 1′-Cu (doublet spin state), 1′-Cd (singlet spin state), 1′-Co (singlet spin state), and 1′-Zn (singlet spin state) are similar, i.e., 27.2, 29.7, 30.5, 30.5, and 31.9 kcal/mol, respectively, and the highest barrier (41.5 kcal/mol) is computed for 1′-Pd (singlet spin state).