Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation.
The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.
Schaertl
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Max-Planck Institut für Biophysikalische Chemie, Abteilung Molekulare Genetik, D-37070 Göttingen, Germany.
Geeves
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MA
Konrad
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000201:enzymology*; D020559:Monomeric GTP-Binding Proteins; D009154:Mutation; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase; D010766:Phosphorylation; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D054778:NM23 Nucleoside Diphosphate Kinases; D014157:Transcription Factors; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C517938:NME1 protein, human; C517940:NME2 protein, human; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins