Novel reactivity of Fhit proteins: catalysts for fluorolysis of nucleoside 5′-phosphoramidates and nucleoside 5′-phosphosulfates to generate nucleoside 5′-phosphorofluoridates

Abstract

Fragile histidine triad (HIT) proteins (Fhits) occur in all eukaryotes but their function is largely unknown. Human Fhit is presumed to function as a tumour suppressor. Previously, we demonstrated that Fhits catalyse hydrolysis of not only dinucleoside triphosphates but also natural adenosine 5'-phosphoramidate (NH2-pA) and adenosine 5'-phosphosulfate (SO4-pA) as well as synthetic adenosine 5'-phosphorofluoridate (F-pA). In the present study, we describe an Fhit-catalysed displacement of the amino group of nucleoside 5'-phosphoramidates (NH2-pNs) or the sulfate moiety of nucleoside 5'-phosphosulfates (SO4-pNs) by fluoride anion. This results in transient accumulation of the corresponding nucleoside 5'-phosphorofluoridates (F-pNs). Substrate specificity and kinetic characterization of the fluorolytic reactions catalysed by the human Fhit and other examples of involvement of fluoride in the biochemistry of nucleotides are described. Among other HIT proteins, human histidine triad nucleotide-binding protein (Hint1) catalysed fluorolysis of NH2-pA 20 times and human Hint2 40 times more slowly than human Fhit.

Adenylylsulfate–ammonia adenylyltransferase activity is another inherent property of Fhit proteins

“…Kinetic parameters of the ammonolytic reactions 3 and 5 were as follows: K m for SO 4 -pA, 2.4±0.3 mM; K m for F-pA, 1.6±0.2 mM; and K m for NH 3 estimated for the ammonolysis of F-pA, 60±15 mM. The hydrolytic reactions 2, 7 and 8 had pH optima between 6.5 and 7 [7,21]. In reactions containing 500 mM ammonium bicarbonate (pH 9), rates were about 20 times slower than at neutral pH, with no reaction proceeding in mixtures containing CAPS and NH 3 at a pH of around 10.6.…”

“…Therefore, these hydrolases await their fourth digit). Very recently, Fhits have been demonstrated to catalyse also fluorolysis of nucleoside 5′-phosphoramidates and nucleoside 5′-phosphosulfates, reactions 9 and 10 [21]. Whether the reported Fhit-mediated ammonolysis of certain nts remains only a curiosity or has some biological relevance requires further studies.…”

Development of Fluorophosphoramidate as a Biocompatibly Transformable Functional Group and its Application as a Phosphate Prodrug for Nucleoside Analogs

“…Specifically, the phosphorus-fluorine (PÀ F) bond [16][17] is known to have high bond energy, and thus shows reasonable resistance against water hydrolysis, which would be the most prevalent reaction under biological conditions. Accordingly, fluorophosphate groups were developed and successfully used in evaluation of enzymatic activity, [18][19][20][21] as NMR-based probes for structure analysis, [22] as potential modifiers for nucleosides [23] and as inositol analogs [24] in medicinal chemistry. In light of these precedents, we had an interest in the amidate analog of fluorophosphate, fluorophosphoramidate (FPA), because amino groups can mitigate the electrophilicity of the phosphorus center to suppress its inherent susceptibility to hydrolysis.…”