Deciphering Differential Distortion of Purine Substrates by Human and Plasmodium falciparum HGPRT

Abstract

Enzymes of the salvage pathway are essential to understanding the biochemistry of parasitic protozoa. These parasites lack de novo pathway for nucleotide synthesis and depend solely on purines salvage from their host. Hypoxanthine guanine phosphoribosyltransferase (HGPRT) from Plasmodium falciparum has long been regarded as a potential therapeutic target. Although structurally homologous to human enzyme, PfHGPRT differs from human (h) HGPRT in its expanded substrate specificity and activity towards common substrates. We used substrate analogues, 9‐deazapurines, in combination with UV resonance Raman spectroscopy to capture non‐covalent interactions in enzyme‐substrate complexes. Raman spectral band positions are sensitive to sub‐angstrom changes in bond length. We observed perturbations in Raman spectra that provide quantitative information about hydrogen bonding and stacking‐interactions with the enzyme active‐site. The bound ligands are extensively distorted towards the transition‐state of phosphoribosyltransferase reaction. Differential loss in Raman crosssection of the ligands bound to two enzymes suggests that active‐site is more compact in hHGPRT than in PfHGPRT. The rigidity of active‐site in hHGPRT is attributed to tighter stacking interaction between phenylalanine residue of the active‐site and the nucleobase.Research was supported by IISER, India and Council for Scientific and Industrial Research, India.