Pig heart nucleosidediphosphate kinase Phosphorylation and interaction with Cibacron blue 3GA

Abstract

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...

Substrate Specificity of Human Nucleoside-diphosphate Kinase Revealed by Transient Kinetic Analysis

“…2). These values are in the same range as determined for D. discoideum NDK of 0.2 (22), for yeast NDK of 0.19 (17), and for pig heart NDK of 0.25 (31).…”

Thermal Stability of Hexameric and Tetrameric Nucleoside Diphosphate Kinases

“…The enzyme concentration was estimated using the following extinction coefficients at 280 nm for 1 mg/ml solutions: 0.6 for the Dictyostelium NDP kinases (Wallet, 1992) and 0.25 for the E. coli enzyme, calculated from the amino acid composition (Gill and von Hippel, 1989). This figure was confirmed by measuring the phosphorylation stoichiometry (Lascu et al, 1983).…”

“…Activity Assay-NDP kinase activity was measured at 25°C by the coupled assay using ATP and 8-bromoinosine 5Ј-diphosphate as donor and acceptor nucleotides as described previously (Lascu et al, 1983). The measure of residual activity in thermal inactivation experiments was performed by quickly diluting the enzyme 100-1000-fold into icecold buffer before further diluting it 100-fold into the assay mixture.…”

The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus

“…ATP hydrolysis assay. The ATP-hydrolyzing activity of purified GSTNla28S-cyt was investigated using a standard colorimetric assay that couples the hydrolysis of ATP with the oxidation of NADH (23). In this assay, the change in absorbance at 340 nm caused by the oxidation of NADH to NAD is used to monitor the amount of ATP hydrolyzed to ADP.…”

“…When they detect a specific signal, HKs bind and hydrolyze ATP and autophosphorylate at a conserved histidine residue (14). To confirm that Nla28S was indeed a functional HK protein, we first examined the in vitro ATPase activity of purified GST-Nla28S-cyt using a colorimetric assay that couples ATP hydrolysis to NADH oxidation (23). As shown in Fig.…”