Pre-steady State of Reaction of Nucleoside Diphosphate Kinase with Anti-HIV Nucleotides

Abstract

The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s ؊1 and a K S of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3-OH in ddNTP causes a 10-fold increase in K D . Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in k cat values.Nucleoside analogues like 3Ј-deoxy-3Ј-azidothymidine (AZT) 1 and dideoxynucleosides (ddN) are widely used as antiviral drugs, particularly in the multitherapy protocols now used in the treatment of AIDS. These drugs are targeted at the HIV reverse transcriptase as the lack of the 3Ј-OH required for the 3Ј-5Ј phosphodiester bond formation during DNA elongation blocks viral DNA synthesis. To exert their antiviral activity, the nucleoside analogues must be phosphorylated into triphosphates derivatives by cellular kinases. Although AZT, ddT, and ddC are structurally related, they show different patterns of intracellular phosphorylation (1). The synthesis of mono-and diphospho-derivatives involves kinases specific for either purines (for example deoxyguanosine kinase) or pyrimidines (for example deoxycytidine kinase or thymidine kinase). In all cases, the last step in the pathway leading to the triphospho-derivative is catalyzed by nucleoside diphosphate (NDP) kinase (EC 2.7.4.6), which has little specificity toward the nucleobase (2).NDP kinase phosphorylates all nucleoside diphosphates into triphosphates using ATP as the major phosphate donor. The reaction involves the formation of a phosphorylated intermediate according to a ping-pong bi-bi mechanism following the scheme below, where E-P is the phosphorylated intermediate on the catalytic histidine (3).EϳP ϩ N 2 DP 7 N 2 TP ϩ E (reaction B) SCHEME IIn recent yea...

Single Strand DNA Specificity Analysis of Human Nucleoside Diphosphate Kinase B

“…The inhibitory effect of CDP is 50-fold lower than that of GDP, demonstrating a base specificity of the inhibition. It is interesting to note that a similar base specificity has also recently been reported for the enzymatic activity of the human NDPK-B (45) and of the Dictyostelium enzyme (46). Moreover, analysis of the inhibition curve in the presence of GTP gives a K I equal to 40 M similar to that of GDP, confirming that formation of the phosphohistidine intermediate does not affect the binding of the oligonucleotide.…”

“…In the filterbinding assay, the oligonucleotide concentration used was near the dissociation constant, conditions in which the stoichiometry could be in error (47). In contrast, a fixed protein concentration and variable oligonucleotide concentrations, such that der et al (46) reported recently that the nucleotide analogue PAPS can adopt a new conformation inside the active site of Dictyostelium NDP kinase. Indeed, the 5Ј-phosphate of bound PAPS points out from the binding pocket, whereas the 3Ј-phosphate binds near the active site histidine.…”

Anti-HIV Host Factor SAMHD1 Regulates Viral Sensitivity to Nucleoside Reverse Transcriptase Inhibitors via Modulation of Cellular Deoxyribonucleoside Triphosphate (dNTP) Levels

“…This result was anticipated based on the previous study (38) and based on our biochemical data showing that increased dNTPs reduce AZT-TP and ddCTP inhibition of RT by active site competition. The reduction in drug efficacy observed in the cell culture experiments may also be influenced by competition for the active site of cellular kinases because NRTIs are not as efficiently phosphorylated as deoxyribonucleosides (33). Additionally, when VLPs ϩVpx were added to activated T cells, NRTI efficacy was decreased to a much lesser extent than VLP ϩVpx-treated macrophages (Fig.…”

“…This suggests that the kinases expressed in macrophages are active enough to phosphorylate a high concentration of deoxyribonucleosides into dNTPs. However, nucleoside diphosphate kinase is not as active toward NRTIs as canonical dNTPs (33). Therefore, when the concentration of dNTPs increases, the dNTPs may out-compete NRTIs for the active site of nucleoside diphosphate kinase, resulting in a decreased concentration of NRTI-TPs.…”

The Enantioselectivity of Enzymes Involved in Current Antiviral Therapy Using Nucleoside Analogues: A New Strategy?

“…The enzyme is independent of the nature of the base and thus displays little specificity with respect to the nucleoside diphosphate and the natural triphosphate donor, although discrimination with respect to 2′,3′-dideoxynucleoside triphosphate donors has been reported (Bourdais et al, 1996;Deville-Bonne et al, 1996;Schneider et al, 1998). The enantioselectivity of the enzyme has not been studied except in the case of beef liver nucleoside diphosphate kinase, which phosphorylates both enantiomers of carbovir with a preference for the natural enantiomer .…”