Aspartate transcarbamoylase (ATCase) catalyzes the second step of the de novo pyrimidine biosynthetic pathway and produces N-carbamoyl-l-aspartate (CA) and inorganic phosphate (Pi) from carbamoyl phosphate (CP) and aspartate (Asp). The ATCase from Trypanosoma cruzi (TcATCase) is a non-allosteric homotrimeric enzyme with all chains being catalytic and weakly inhibited by the general ATCase inhibitor, N-phosphonoacetyl-l-aspartate (PALA). While TcATCases is a compelling drug target, the lack of detailed structural insights into its reaction mechanism hinders the design of an effective effector. In this study X-ray crystal structure analyses of TcATCase in ligand-free, CP- and PALA-bound forms were carried out up to about 2.0 Å resolution. The determined crystal structures show that the ligand-free chain and chains binding CP and Asp, CA and Pi, Pi, Asp and Pi, and Asp were captured in the crystals. Based on these structures, we present the detailed mechanism of the TcATCase catalysis described as the ordered Bi–Bi mechanism. These findings provide a detailed catalytic snapshots of the ATCase obtained in the crystalline state, including directly captured reaction intermediates, and offer new structural insights suggesting ordered Bi–Bi mechanism for TcATCase. The captured structure of the TcATCase-PALA complex will accelerate the design of PALA-based new anti-trypanosomal drug.
Matoba et al. (Thu,) studied this question.