Estudio de las actividades y los centros activos de las enzimas ADPRibas-Mn y trioquinasa/FMN ciclasa

Laburpena

The activities and the active centers of Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn) and triokinase/FMN cyclase (TKFC), both obtaines as recombinant proteins, were investigated. Concerning ADPRibase-Mn, the enzymes from rat (�Rattus norvegicus�) and zebrafish (�Danio rerio�) were studied. For the first time, structural and biochemical information were put together for the same protein of the ADPRibase-Mn family. Novel ADPRibase-Mn activities were found, namely cyclic ADP-ribose phosphohydrolase and 2´,3´-cyclic nucleotide phosphodiesterase. The active centers of both ADPRibase-Mn enzymes were defined from the structure of the �D. rerio� protein and by molecular modeling. A molecule of water and a histidine residue well positioned for the hydrolytic reaction were identified. The role of such residue was confirmed by mutagenesis. The study of TKFC was centered on the human enzyme (hTKFC), a homodimer of two-domain (K and L) subunits. hTKFC models bound to kinase substrates, cyclase substrate, or without substrates, were constructed. Molecular dynamics simulations revealed movements of the K domains necessary for the kinase activity. Molecular modeling led to the identification of relevant interactions of several aminoacids. Biochemical studies performed with hTKFC included the demonstration that individual K and L domains are devoid of kinase activity, while K alone is active as FMN cyclase. In addition, six point mutants were prepared and, together with another previously prepared one, gave relevant information on the active center and the role of both domains in catalysis.