Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase.
Nucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000-fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.
Schneider
Benoit
B
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS FRE2364, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Norda
Ameli
A
Karlsson
Anna
A
Veron
Michel
M
Deville-Bonne
Dominique
D
D019943:Amino Acid Substitution; D000818:Animals; D004023:Dictyostelium; D005453:Fluorescence; D007525:Isoelectric Focusing; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism
D009711:Nucleotides; D010710:Phosphates; D009701:Nucleoside-Diphosphate Kinase; C035638:thiophosphoric acid