AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug-resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH(3)(-)) group on the alpha-phosphate, and found that they are substrates for both enzymes. X-ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH.O bond contributing to catalysis, and the R(p) diastereoisomer of thymidine alpha-boranotriphosphate bound like a normal substrate. Using alpha-(R(p))-boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the alpha-borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug-resistant viruses. Thus, the alpha-borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.

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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...

The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a phosphorylated product proceeds via a dissociative transition state with little bond formation to the nucleophile. Consideration of the dissociative nature of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl transfer? We have addressed this question for the NDP kinase reaction. A mutant form of the enzyme lacking the nucleophilic histidine (H122G) can be chemically rescued for ATP attack by imidazole or other exogenous small nucleophiles. The ATP reaction is 50-fold faster with the wild-type enzyme, which has an imidazole nucleophile positioned for reaction by a covalent bond, than with H122G, which employs a noncovalently bound imidazole nucleophile [(kcat/KM)ATP]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from comparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole and water toward ATP in solution. X-ray structural analysis shows no detectable rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of approximately 10(2)-fold for the covalent imidazole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is underscored by the more deleterious effect of replacing ATP with AlphaTauPgammaS in the wild-type reaction than in the imidazole-rescued mutant reaction, as follows. For the wild-type, AlphaTauPgammaS presumably disrupts positioning between nucleophile and substrate, resulting in a large thio effect of 300-fold, whereas precise alignment is already disrupted in the mutant because there is no covalent bond to the nucleophile, resulting in a smaller thio effect of 10-fold. In summary, the results suggest a catalytic role for activation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.