Phosphorylation of pyrimidine L-deoxynucleoside analog diphosphates. Kinetics of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by 3-phosphoglycerate kinase.
Anticancer and antiviral D- and L-nucleoside analogs are phosphorylated stepwise in the cells to the pharmacologically active triphosphate metabolites. We recently reported that in the last step, L-deoxynucleoside analog diphosphates are phosphorylated by 3-phosphoglycerate kinase (PGK). To explain the preference of PGK for L- over D-deoxynucleoside analog diphosphates, the kinetics of their phosphorylation were compared with the dephosphorylation of the respective triphosphates using recombinant human PGK. The results attributed favorable phosphorylation of L-deoxynucleoside analog diphosphates by PGK to differences in k(cat), which were consequences of varied orientations of the sugar and diphosphates in the catalytic site of PGK. The amino acids involved in the catalytic reaction of PGK (including Glu(344), Lys(220), and Asn(337)) were therefore mutated. The impact of mutations on the phosphorylation of L- and D-deoxynucleoside analog diphosphates was different from those on dephosphorylation of the respective triphosphates. This suggested that the interactions of the nucleoside analogs with amino acids during the transition state are different in the phosphorylation and dephosphorylation reactions. Thus, reversible action of the enzyme may not involve the same configuration of the active site. Furthermore, the amino acid determinants of the action of PGK for L-deoxynucleotides were not the same as for the D-deoxynucleotides. This study also suggests the potential impact of nucleoside analog diphosphates and triphosphates on the multiple cellular functions of PGK, which may contribute to the action of the analogs.
Krishnan
Preethi
P
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Liou
Jieh-Yuan
JY
Cheng
Yung-Chi
YC
D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000493:pharmacokinetics; D000998:Antiviral Agents / Q000493:pharmacokinetics; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D008961:Models, Structural; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011684:Purine Nucleosides / Q000493:pharmacokinetics*; D011685:Purine Nucleotides / Q000493:pharmacokinetics*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D017931:DNA Primers; D011684:Purine Nucleosides; D011685:Purine Nucleotides; D011994:Recombinant Proteins; D010735:Phosphoglycerate Kinase