Catalytic Mechanism and Allosteric Regulation of UDP-Glucose Pyrophosphorylase from Leishmania major

UDP-glucose pyrophosphorylase (UGP) is a nucleotidyltransferase of central importance in all organisms and considered an attractive drug target in the human pathogens Leishmania and Trypanosoma. Here, we used wild-type and mutant Leishmania major UGP to solve the crystal structures of postreactive, UTP, and UDP-Glc bound states and performed kinetic and theoretical chemistry analysis of the enzymatic reaction. The new data filled critical gaps in the knowledge of the UGP mechanism and allowed reconstructing the complete enzymatic cycle on three levels: global (movements of molecular functional blocks), local (behavior of separate residues), and chemical (quantum mechanical description of enzymatic reaction). Results were integrated into a model of UGP activity describing structural changes along the cycle, the mechanisms of substrate binding, UGP catalysis, and product release. Our study revealed the mechanisms of allosteric regulation common for nucleotidyltransferases and, in particular, the mechanical control of the chemical reaction in the active site.