Human nucleoside diphosphate kinase B (Nm23-82) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation

The Se-122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/ Nm23-H2 was recently found in melanoma cells. In comparison to the wild-typ e enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to an alyze phosphoryl transfer between the mutant enzyme and various pairs of nu cleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine resid ue (His(118)) were studied separately by making use of the intrinsic fluore scence changes which occur during these reactions. All apparent second orde r rate constants are drastically reduced, falling 3-fold for phosphorylatio n and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold re duced compared with the wild-type. Thus, the rate-limiting step of the NDK- B-S122P-catalyzed reaction is phosphoryl transfer from the phospho enzyme i ntermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type pr otein. This results in a pronounced shift of the equilibrium between unphos phorylated and phosphorylated enzyme, Moreover, like the Killer-of-prune mu tation in Drosophila NDK and the neuroblastoma Ser(120) --> Gly mutation in human NDK-A/Nm23-H1, the Ser(122) --> Pro substitution in NDK-B affects th e stability of the protein toward heat and urea. These significantly altere d properties may be relevant to the role of the mutant enzyme in various in tracellular processes.