tailieunhanh - Báo cáo khoa học: Active and regulatory sites of cytosolic 5¢-nucleotidase

Cytosolic 5¢-nucleotidase (cN-II), which acts preferentially on 6-hydroxypu-rine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. | Active and regulatory sites of cytosolic 5 -nucleotidase Rossana Pesi1 Simone Allegrini2 Maria Giovanna Careddu1 2 Daniela Nicole Filoni1 Marcella Camici1 and Maria Grazia Tozzi1 1 Dipartimento di Biologia Units di Biochimica Universita di Pisa Pisa Italy 2 Dipartimento di Scienze delFarmaco University di Sassari Sassari Italy Keywords cN-II active site cN-II regulatory sites cN-II structure cytosolic 5 -nucleotidase II Correspondence M. G. Tozzi Dipartimento di Biologia Via S. Zeno 51 Pisa Italy Fax 39 502211450 Tel 39 502211457 E-mail mtozzi@ Received 19 July 2010 revised 10 September 2010 accepted 21 September 2010 doi Cytosolic 5 -nucleotidase cN-II which acts preferentially on 6-hydroxypu-rine nucleotides is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phosphointermediate. Both activities are regulated by a number of phosphorylated compounds such as diadenosine tetraphosphate Ap4A ADP ATP 2 3-bisphosphoglycerate BPG and phosphate. On the basis of a partial crystal structure of cN-II we mutated two residues located in the active site Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phosphointermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site R144 N154 I152 and three in the second F127 M436 and H428 and found that Ap4A and ADP interact with the same site but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from .