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

Abstract

Nucleoside-diphosphate kinases (NDKs) catalyze the transfer of ␥-phosphoryl groups from NTPs via an active site histidine to NDPs using a ping-pong mechanism. We have used the change of intrinsic tryptophan fluorescence that occurs upon phosphorylation of NDK to measure the rates of phosphorylation and dephosphorylation with a range of nucleotides and nucleotide analogues. For natural nucleotides, the rates of phosphorylation and dephosphorylation were linearly dependent upon nucleotide concentration until they became too fast to measure. The second order rate constants for phosphorylation by natural NTPs varied between 0.7 and 13 ؋ 10 6 M ؊1 s ؊1 . Dephosphorylation by NDPs was 2-3-fold faster than the corresponding phosphorylation reaction, and dephosphorylation by dNDPs was 3-4-fold slower than the equivalent NDPs. In all cases, second order rate constants were highest for guanine followed by adenine and lowest for cytosine nucleotides. NDK also catalyzes the transfer of thiophosphate from adenosine 5-O-(thiotriphosphate) (ATP␥S) and guanosine 5-O-(thiotriphosphate) (GTP␥S) to NDP, but at 1 ⁄1000 of the equivalent phosphoryl transfer rates. In this case, the observed rate constants of phosphorylation and dephosphorylation were hyperbolically dependent on nucleotide concentration. Thiophosphorylation by ATP␥S and GTP␥S occurred with k max of 2.8 and 1.35 s ؊1 and K d of 145 and 36 M respectively. For dethiophosphorylation by a range of NDPs, k max was in the range of 5-30 s ؊1 , whereas K d varied between 0.16 and 3.3 mM. Guanine had the lowest K d values, and cytosine had the highest. The data are consistent with fast reversible binding of the nucleotide followed by the rate-limiting phosphoryl transfer. Thiophosphates change only the rate of the phosphoryl transfer step, whereas both events are influenced by the base. Modification at the 2-hydroxyl of ribose has only a small effect, while the overall rate of phosphoryl transfer is reduced 1000-fold by modification at the 3-ribose.Nucleoside-diphosphate kinase (NDK 1 ; EC 2.7.4.6) is a ubiquitous enzyme that catalyzes the transfer of the ␥-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate (1), involving a phosphohistidine intermediate state (2). NDK exists as either hexamers (most eukaryotic species) or tetramers (most prokaryotic species) and has been reported to display little specificity for different nucleotide bases (2, 3), reflecting a pivotal role in maintaining balanced levels of all oxy-and deoxynucleoside triphosphates in the cell. It is thus considered a housekeeping enzyme (4), a role that has taken on greater significance with the therapeutic use of nucleotide analogues such as azidothymidine (AZT) as replication inhibitors as these compounds must be phosphorylated in the cell before becoming active (5).In humans, two very closely (89% identity) related isoforms of NDK, designated NDK-A and NDK-B, were identified biochemically (6), which associate in vivo to form mixed hexameric isoenzymes. Evidence that NDK-A and -B may...

Human Nucleoside Diphosphate Kinase B (Nm23-H2) from Melanoma Cells Shows Altered Phosphoryl Transfer Activity Due to the S122P Mutation

“…Rapid Kinetics-The rates of nucleotide-induced protein fluorescence changes at 20°C in buffer A were measured with a Hi-Tech Scientific SF-51 stopped-flow apparatus equipped with a 100-watt xenon/mercury lamp and monochromator as described before (14). Intrinsic tryptophan fluorescence was excited at 295 nm and monitored through a 320-nm long-pass filter.…”

“…The NDK-B proteins carrying the N-terminal histidine-tag (His-tag) were purified on a nickel-agarose column (Qiagen) according to the instructions of the supplier. The 21-amino acid N-terminal extension was shown to have no influence on the activity of the enzyme (14). Mutant NDK-A was purified like wild-type NDK-A as described before (9).…”

“…Mutagenesis and Cloning-All reagents were obtained from Sigma and Roche Molecular Biochemicals, unless stated otherwise, and treated as described before (14). The mutations S122P and S120G were introduced by site-directed mutagenesis after the Kunkel Method (15) using the Muta-Gene kit (Bio-Rad) and the mutagenic primers: 5Ј-CATGGCAGTGATCCGGTAAAAAGTGCTG-3Ј for S122P and 5Ј-GAA-CATTATACATGGCGGTGATTCTGT-3Ј for S120G (9).…”

“…The titration experiments to determine K eq were performed as described before (14), using ATP as phosphoryl donor. At half-saturation of the fluorescence change, the following relations apply: …”

“…The effects of mutations leading to metastatic tumor cell lines are not clear, even though there is evidence from Drosophila NDK mutants that stability of the oligomeric enzyme may play a major role (11). In the present study we applied transient kinetic methods to examine the phosphoryl transfer reactions of the mutants NDK-B S122P and NDK-A S120G , an approach we successfully used before to characterize human wild-type NDKs (14). We measured the phosphorylation and the dephosphorylation reactions of both mutant enzymes with different substrates and compared them with the wild-type enzymes under identical experimental conditions.…”