The glutamine amidotransferase (GAT) family of enzymes produce and utilize ammonia in vital biochemical pathways. The bienzyme imidazole glycerol phosphate synthase (IGPS) is a GAT composed of glutaminase and cylcase subunits that perform the hydrolysis of glutamine to form glutamate and ammonia, which goes on to react with the allosteric ligand (PrFAR) 20 Å away in the cyclase active site. The catalytic rate of IGPS is critically dependent on allosteric activation. Binding of PrFar results in a 4900-fold increase in activity. The mechanism of allosteric regulation in IGPS has received intense interest, revealing how the two active sites are connected through residue pathways, however there is no clear answer of how the glutaminase activity changes upon effector binding. To answer this question we performed quantum mechanical calculations to investigate the hydrolytic mechanism in the glutaminase active site of IGPS on two reduced active site models initiated from crystal structures where the effector molecule is and is not bound. The models are composed of a catalytic triad (Cys-His-Glu) that is characteristic of class-I GATs. Additional residues were included based on involvement with the glutamine substrate. Evaluation of the glutaminase reaction profiles when PrFAR is and is not bound reveal the structural and energetic consequences for the glutaminase active site as a result of allosteric activation. Understanding the changes that can occur in an enzyme active site as a consequence of effector binding is a rudimentary step in being able to artificially design active sites that can be modulated by allosteric perturbation.

Quantum mechanical modeling of IGPS glutaminase reaction mechanism to reveal method of allosteric activation

ACS Fall 2020 Virtual Meeting

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