Abstract
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.
Highlights
Anticancer and antiviral D- and L-nucleoside analogs are phosphorylated stepwise in the cells to the pharmacologically active triphosphate metabolites
This study suggests the potential impact of nucleoside analog diphosphates and triphosphates on the multiple cellular functions of phosphoglycerate kinase (PGK), which may contribute to the action of the analogs
Through comparison of phosphorylation of several clinically relevant D-deoxynucleoside, D-dideoxynucleoside, and L-deoxynucleoside analog diphosphates by nucleoside-metabolizing enzymes purified from the human hepatic carcinoma cell line HepG2, we recently reported that L-deoxynucleoside analog diphosphates could be phosphorylated by 3-phosphoglycerate kinase (PGK); D-deoxynucleoside analog diphosphates were likely to be phosphorylated by nucleoside diphosphate kinase; and D-dideoxynucleoside analog diphosphates were excellent substrates for creatine kinase [16]
Summary
L-SddC, -L-2Ј,3Ј-dideoxy-3Ј-thiacytidine; L-FMAU, 2Ј-fluoro-5-methyl--L-arabinofuranosyluracil; L-ddC, -L-2Ј,3Ј-dideoxycytidine; HIV, human immunodeficiency virus; PGK, 3-phosphoglycerate kinase; DP, diphosphate; TP, triphosphate; HPLC, high pressure liquid chromatography; AMP-PNP, adenosine 5Ј(,␥-imino)triphosphate. ATP and 3-phosphoglycerate could inhibit reduction of plasmin, presumably through inducing a conformational change that was not favorable to the reduction of plasmin [25] This suggested that the level of nucleoside diphosphates or triphosphates (natural or otherwise) might have a regulatory impact on the multiple cellular functions of PGK. The preference observed was further explained through kinetic analysis of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by single amino acid mutants of PGK. These included mutations of PGK at Glu344, Lys220, and Asn337, all of which are involved in hydrogen bonding with the sugar and phosphate side chain of nucleoside analog diphosphates or triphosphates during the catalytic reaction
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