Phosphorylation of nucleoside diphosphate kinase at the active site studied by steady-state and time-resolved fluorescence.
Nucleoside diphosphate (NDP) kinase is the enzyme responsible in the cell for the phosphorylation of nucleoside or deoxynucleoside diphosphates into the corresponding triphosphates at the expense of ATP. Transfer of the gamma-phosphate is very fast (turnover number above 1000 s-1) and involves the phosphorylation of a histidine residue at the active site of the enzyme. We have used intrinsic protein fluorescence of the single tryptophan of Dictyostelium discoideum NDP kinase as a sensitive probe for monitoring the interaction of the enzyme with its substrates. We demonstrate that the 20% quenching of steady-state fluorescence observed upon addition of ATP is due to formation of the phosphorylated intermediate. Time-resolved fluorescence indicates that the Trp-137 side chain is rigidly bound to the protein core with a unique lifetime of 4.5 ns for the free enzyme at 20 degrees C and that it remains tightly immobilized during the time course of the reaction. Phosphorylation of this catalytic residue (His-122) in the presence of ATP induces a similar decrease in mean lifetime, due to the splitting of the signal and the appearance of a shorter decay. This splitting is discussed in terms of a slow conformational equilibrium. We demonstrate that, in the wild-type enzyme, the conserved His-55 quenches the fluorescence of Trp-137 as the H55A mutant protein fluorescence displays an increase in quantum yield. Even though H55A mutant enzyme is active, the absence of the imidazole ring prevents the detection of the phosphorylated state of His-122 by Trp-137. We conclude that His-55 serves as a relay between His-122 and Trp-137.
Deville-Bonne
D
D
Laboratoire de Modélisation et d'Ingéniérie des Protéines, Université Paris-Sud Orsay, France.
Sellam
O
O
Merola
F
F
Lascu
I
I
Desmadril
M
M
Véron
M
M
D000818:Animals; D001665:Binding Sites; D004023:Dictyostelium; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence
D009701:Nucleoside-Diphosphate Kinase