title: Catalytic and regulatory roles of species involved in metal-nucleotide equilibriums in human pyridoxal kinase.
abstract: Pyridoxal 5'-phosphate is the active form of vitamin B6 and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we analyse the catalytic and regulatory mechanisms of hPLK as a function of a precise determination of the species involved in metal-nucleotide equilibriums and describe new regulatory mechanisms for this enzyme. hPLK activity is supported by several metals, being Zn(2+) the most effective, although the magnitude of the effect observed is highly dependent on the relative concentrations of metal and nucleotide used. The true substrate for the reaction catalyzed by hPLK is the metal nucleotide complex, while ATP(4-) and HATP(3-) did not affect the activity. The enzyme presents substrate inhibition by both pyridoxal (PL) and ZnATP(2-), although the latter behaves as a weakly inhibitor. Our study also established, for the first time, a dual role for free Zn(2+); as an activator at low concentrations (19 μM optimal concentration) and as a potent inhibitor with a IC50 of 37 μM. These results highlighted the importance of an accurate estimation of the actual concentration of the species involved in metal-nucleotide equilibriums in order to obtain reliable values for the kinetic parameters, and for determine the true regulators of the PLK activity. They also help to explain the dissimilar kinetic parameters reported in the literature for this enzyme.
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
authors.forename: Freddy
authors.initials: F
authors.lastname: Navarro
authors.forename: César A
authors.initials: CA
authors.lastname: Ramírez-Sarmiento
authors.forename: Victoria
authors.initials: V
authors.lastname: Guixé
mesh_terms: D055162:Biocatalysis; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011731:Pyridoxal Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D015032:Zinc / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D009711:Nucleotides; D011731:Pyridoxal Kinase; D015032:Zinc