Conformational Changes and Free Energy Landscape of the Unbinding of External Aldimine at the Active Site of Ornithine Decarboxylase in Aqueous Medium

The pyridoxal-5'-phosphate (PLP)-dependent enzymes constitute an important class of enzymes that undergo crucial conformational changes between their apo and holo forms, which are essential for their functional versatility. In this study, we have investigated the conformational changes of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, and the unbinding of the PLP-substrate complex (external aldimine) at the active site of the enzyme using molecular dynamics and well-tempered metadynamic simulations. The study reveals a three-step mechanism for the ligand unbinding process. Initially, the enzyme undergoes a reorganization in which the distance between the C-terminal and N-terminal domains of opposite chains that form the active site increases. This reorganization opens the active site, and the interactions between the PLP-substrate complex and two active site loops, namely loop1 and loop2, begin to weaken. As a result, the ligand exits the active site and primarily interacts with loop3. Over time, these interactions with loop3 also weaken, and the ligand eventually becomes fully unbound. The calculated free energy differences for these steps are found to be 1 kcal/mol, 6 kcal/mol, and 8 kcal/mol, respectively. Our findings provide detailed insights into the conformational changes and energetics associated with ligand unbinding in ODC, offering a valuable framework for understanding similar processes in other PLP-dependent enzymes.