Glycinamide ribonucleotide transformylase (GAR Tfase) is a regulatory enzyme in the de novo purine biosynthesis which has been shown to be an anticancer target. Many drugs have been designed considering the GAR Tfase enzyme as a target to prevent the division of cancerous cells. E. coli GAR Tfase displays pH-dependent conformational changes and ligand binding which has been linked to its catalytic activity. The pH-activity curve gives information about the number of residues or the group of residues involved in the catalysis. However, over-interpretation of the pH-activity curve and use of low-resolution x-ray structures to assign hydrogen bond interactions lead ambiguity in assigning the acid and base catalysts and its catalytic mechanism. We have used the pH-replica exchange molecular dynamics (pH-REMD) simulations implemented in the AMBER package to study the pH-dependent functions such as conformational changes and the binding of ligands. We have used the Wyman-Tanford relation to compute the free energy of the ligand binding with respect to pH. We filtered out residues in their specific protonation states which keeps the structure intact and keeps the ligands bound to the enzyme. We have reproduced the pH-activity curve computationally; the half pKa values along with its shape match very well with the experimental pH-activity curve. This study allows us to assign the general acid and base catalysts unambiguously and using this information, we proposed a novel catalytic mechanism for the E. coli GAR Tfase enzyme. Furthermore, we have found that pH alters the binding of both ligands and the binding of one ligand pre-organizes the active site for another ligand and increases its binding affinity with respect to pH.

Study of the pH-dependent properties for the Escherichia coli glycinamide ribonucleotide transformylase using theoretical methods

2019 Spring National Meeting

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