title: Kinetic mechanism of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.
abstract: Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) exhibits a kinetic mechanism that differs from that of the human homolog. Human HGPRT follows a steady-state ordered mechanism, wherein PRPP binding precedes the binding of hypoxanthine/guanine and release of product IMP/GMP is the rate limiting step. In the current study, initial velocity kinetics with PfHGXPRT indicates a steady-state ordered mechanism, wherein xanthine binding is conditional to the binding of PRPP. The value of the rate constant for IMP dissociation is greater by 183-fold than the kcat for hypoxanthine phosphoribosylation and this results in the absence of burst in progress curves from pre-steady-state kinetics. Further, IMP binding is 1000 times faster (4s(-1) at 0.5μM IMP) when compared to the kcat (3.9±0.2×10(-3)s(-1)) for the reverse IMP pyrophosphorolysis reaction. These results lend support to the fact that in both forward and reverse reactions, the process of chemical conversion (formation of IMP/hypoxanthine) is slow and the events of ligand association and dissociation are faster.
authors.affiliation: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India.
authors.forename: Sourav
authors.initials: S
authors.lastname: Roy
authors.affiliation: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India.
authors.forename: Lakshmeesha K
authors.initials: LK
authors.lastname: Nagappa
authors.affiliation: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India.
authors.forename: Vasudeva S
authors.initials: VS
authors.lastname: Prahladarao
authors.affiliation: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India. Electronic address: hb@jncasr.ac.in.
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Balaram
mesh_terms: D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D016778:Malaria, Falciparum / Q000469:parasitology*; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019820:Xanthine / Q000737:chemistry / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D019820:Xanthine; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase
keywords: IMP pyrophosphorolysis; Kinetic mechanism; PfHGXPRT; Pre-steady-state kinetics; Single-turnover