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The nucleosidediphosphate kinase phosphorylation reaction led to the incorporation of 0.95 i 0.1 phosphate groups per enzyme subunit. The equilibrium constant of the phosphorylation reaction was 0.26. The inhibition of the nucleosidediphosphate kinase activity by Cibacron bluc 3GA was competitive with respect to ATP, the donor nucleotide (apparent Ki = 0.28 pM) and uncompetitive with respect to 8-bromoinosine 5'-diphosphate, the acceptor nucleotide (apparent Ki = 0.31 pM). By difference spectroscopy it was shown that each enzyme subunit bound one Cibacron blue 3GA molecule, whereas the phosphorylated enzyme had no affinity for the dye. ATP was an effective competitor, being able to displace the dye from its bound state. The complex behaviour noted was taken as evidence for cooperative interaction between the enzyme subunits. The data obtained using polarographic techniques agreed with these results.The nucleosidediphosphate (NDP) kinase is a key enzyme in the rephosphorylation of non-adenine nucleoside diphosphates to the corresponding nucleoside triphosphates. The enzyme is built up of six subunits of relatively small size [I -31. The NDP kinases of various origins have a pingpong mechanism. The covalent binding of a phosphate group to a histidine side-chain was noted in all cases [4,5]. Fast kinetic studies [6] and the retention of transfered phosphate group configuration [7] indicate that the phosphorylated enzyme is the true intermediate during the catalysis. The data on the stoichiometry of phosphorylation are conflicting. On the other hand, many structural and mechanistic details are still unknown, such as the location and number of nucleotide binding sites, the interactions among subunits, the role of the conformational transitions, etc.The polysulfonated anthraquinonic dye Cibacron blue 3GA (Reactive Blue 2, Colour Index constitution number 61 21 1) is extensively used as a probe for nucleotide binding sites in enzymes [8 -101. The investigation of the interaction of this dye with N D P kinase is interesting for sevcral reasons. First of all the enzyme binds strongly to blue-Sepharose [1,1 I] and to blue-dextran-Sepharose [12], a property which enabled its purification. Next, given the existence of the phosphorylated enzyme, the use of Cibacron blue 3GA allows the probing (or mapping) of the nucleotide binding site. Finally, if it is true that in spite of known limitations [I31 the dye binds preferentially to the supersecondary structure, called 'dinucleotide fold' [I 4,151, the interaction between Cibacron blue 3GA and N D P kinase might supply information on this functional domain before the three-dimensional structure of the enzyme is elucidated by X-ray diffraction methods.In this study we investigated the interaction of Cibacron blue 3GA with the unphosphorylated and phosphorylated NDP kinase, by steady-state kinetics, difference spectroscopy and polarographic methods. MATERIALS A N D METHODS ChemicalsAll natural nucleotides, substrates and coupling enzymes were from Boehringer (Mannheim). 8-Bromoi...

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The binding of nucleotides to pig heart nucleoside-diphosphate kinase was studied using Rose Bengal as an optical probe. ATP, in the absence of Mg 2+ binds slowly to the enzyme, with a second order rate con-, stant of about 3000 M-W', whereas in its presence the binding is much faster. This finding suggests the regulation of the nucleoside-diphosphate kinase activity by uncomplexed ATP, and that ATP binds normally to the enzyme via a metal ion bridge.