title: Differential Distortion of Purine Substrates by Human and Plasmodium falciparum Hypoxanthine-Guanine Phosphoribosyltransferase to Catalyse the Formation of Mononucleotides.
abstract: Plasmodium falciparum (Pf) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential therapeutic target. Compared to structurally homologous human enzymes, it has expanded substrate specificity. In this study, 9-deazapurines are used as in situ probes of the active sites of human and Pf HGPRTs. Through the use of these probes it is found that non-covalent interactions stabilise the pre-transition state of the HGPRT-catalysed reaction. Vibrational spectra reveal that the bound substrates are extensively distorted, the carbonyl bond of nucleobase moiety is weakened and the substrate is destabilised along the reaction coordinate. Raman shifts of the human and Pf enzymes are used to quantify the differing degrees of hydrogen bonding in the homologues. A decreased Raman cross-section in enzyme-bound 9-deazaguanine (9DAG) shows that the phenylalanine residue (Phe186 in human and Phe197 in Pf) of HGPRT stacks with the nucleobase. Differential loss of the Raman cross-section suggests that the active site is more compact in human HGPRT as compared to the Pf enzyme, and is more so in the phosphoribosyl pyrophosphate (PRPP) complex 9DAG-PRPP-HGPRT than in 9-deazahypoxanthine (9DAH)-PRPP-HGPRT.
authors.affiliation: Indian Institute of Science Education and Research, Pune-411008 (India).
authors.forename: Vishakha
authors.initials: V
authors.lastname: Karnawat
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore-560065 (India).
authors.forename: Spriha
authors.initials: S
authors.lastname: Gogia
authors.affiliation: Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore-560064 (India). hb@jncasr.ac.in.
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Balaram
authors.affiliation: Indian Institute of Science Education and Research, Pune-411008 (India). mrinalini@iiserpune.ac.in.
authors.forename: Mrinalini
authors.initials: M
authors.lastname: Puranik
mesh_terms: D055162:Biocatalysis; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism*; D008958:Models, Molecular; D009711:Nucleotides / Q000096:biosynthesis* / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D010963:Plasmodium falciparum / Q000201:enzymology*; D011687:Purines / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009711:Nucleotides; D011687:Purines; D010649:Phenylalanine; D006147:Guanine; C076777:9-deazaguanine; D007041:Hypoxanthine Phosphoribosyltransferase
keywords: density functional theory; resonance Raman spectroscopy; substrate analogue; substrate distortion; transition state