Orotidine 5?-monophosphate decarboxylase (ODCase) catalyzes the conversion of orotidine-5?-phosphate (OMP) to uridine 5?-phosphate (UMP) in the last step of de novo pyrimidine biosynthesis. As a key participant in an important biosynthetic pathway, ODCase is a target for inhibition by potential anti-cancer agents. Understanding the nature of the mechanism and the transition state in the enzyme-catalyzed reaction is essential for the advancement of inhibitor design. Although a wealth of experimental and theoretical studies have been performed on ODCase, the mechanism of this enzyme is not clearly understood and is currently at the center of an ongoing controversy. This study aims to solve the mechanistic puzzle of ODCase through a unique collaboration between theory and experiment. Advanced theoretical techniques, including free energy perturbation methods, combined quantum mechanics and molecular mechanics, and density functional theory, will be employed in a thorough investigation of ODCase that will directly parallel experimental studies. Specifically, these methods will be used to determine the binding orientation of OMP and other substrates/inhibitors to the enzyme, generate free energy profiles for potential mechanisms, and analyze transition state structures through the calculation of secondary isotope effects.
