The catalytic effects of active site conformational change in the allosteric activation of imidazole glycerol phosphate synthase

Imidazole glycerol phosphate synthase (IGPS) is a heterodimeric class-I glutamine amidotransferase (GAT) that hydrolyzes glutamine. Ammonia is produced and transferred to a second active site where it reacts with N1-(5'- phosphoribosyl)-formimino-5-aminoimidazole-4 carboxamide ribonucleotide (PrFAR) to form precursors to purine and histidine biosynthesis. Binding of PrFAR over 25 Å away from the active site increases glutaminase efficiency by ~4500-fold, primarily altering the glutamine turnover number. IGPS has been the focus of many studies on allosteric communication; however, atomic details for how the glutamine hydrolysis rate increases in the presence of PrFAR are lacking. We present a density functional theory study on 237-atom active site cluster models of IGPS based on crystallized structures representing the inactive and allosterically-active conformations and investigate the multistep reaction leading to thioester formation and ammonia production. The proposed mechanism is supported by similar, well-studied enzyme mechanisms, and the corresponding energy profile is consistent with steady-state kinetic studies of PrFAR+IGPS. Additional active site models are constructed to examine the relationship between active site structural change and transition state stabilization via energy decomposition schemes. The results reveal that the inactive IGPS conformation does not provide an adequately formed oxyanion hole structure and that repositioning of the oxyanion strand relative to the substrate is vital for a catalysis-competent oxyanion hole, with or without the hVal51 dihedral flip. These findings are valuable for future endeavors in modeling the IGPS allosteric mechanism by providing insight into the atomistic changes required for rate enhancement that can inform suitable reaction coordinates for subsequent investigations.