Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3-6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the K(m) for Ap3A and Ap4A are 1.3 and 4.6 microM, respectively. Values of the specificity constant, kcat/K(m), for Ap3A and Ap4A are 2.0 x 10(6) and 6.7 x 10(3) s-1 M-1, respectively, for a glutathione S-transferase (GST)-Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Also, Fhit is the first HIT protein in which the histidine residues have been demonstrated by mutagenesis to be critical for function.

J. Biol. Chem. 274, 8604 -8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa-and pentaphosphates (Ap 6 A and Ap 5 A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap 6 A and Ap 5 A, in preference to other diadenosine polyphosphates. The emergence of Ap 6 A and Ap 5 A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process.Following the discovery of dinucleoside polyphosphates in biological systems over 30 years ago (1), these compounds have been studied extensively in prokaryotic and eukaryotic organisms. Several important intracellular and extracellular signaling functions have now been ascribed to the diadenosine compounds, Ap 3 A, 1 Ap 4 A, Ap 5 A, and Ap 6 A (2-4). Indeed, the ultimate fate of cell lineages and the very survival of an organism may depend upon the tight control of cellular diadenosine polyphosphate metabolism. For example, the intracellular level of Ap 4 A has long been known to be associated with cell proliferation (5). Moreover, it was recently proposed (6) that Ap 3 A has an antiproliferative role when complexed with the putative tumor suppressor Fhit protein, an Ap 3 A hydrolase (7,8). Thus, the Ap 3 A/Ap 4 A ratio may be an important factor in determining the alternative cellular fates of proliferation, differentiation, and apoptosis (3, 9). In higher eukaryotes, Ap n A appear also to be intracellular mediators of certain extracellular stimuli; they respond to glucose in pancreatic ␤-cells (10). Ap n A may also regulate ATP-sensitive K ϩ channels in ␤-cells and cardiac muscle (11-13) and intracellular ryanodine-binding Ca 2ϩ -release channels in cardiac and skeletal muscle, and in the brain (14, 15). Finally, extracellular Ap 5 A and Ap 6 A have also been identified as neurotransmitters (2) and vasomodulators (16,17).In addition to these physiological functions, Ap n A respond to heat shock and oxidative stress with an increase in concentration (18). If allowed to accumulate, they could prove toxic through their ability to inhibit nucleotide kinases (19,20), protein kinases (21,22), and other enzymes (23). Thus, there is considerable interest in the enzymes t...

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Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (Ap(n)A, n =5 or 6) and diphosphoinositol polyphosphates [diphosphoinositol pentakisphosphate (PP-InsP(5)) and bisdiphosphoinositol tetrakisphosphate ([PP](2)-InsP(4))] in vitro. The in vivo substrates of Aps1 are unknown. We report here the identification of Ap(5)A, PP-InsP(5), [PP](2)-InsP(4) and a novel diphosphoinositol polyphosphate ([PP](x)-InsP(x)) in S. pombe using HPLC methods. Ap(5)A was present at 0.06 pmol/mg of protein (approx. 4 nM). PP-InsP(5), [PP](x)-InsP(x) and [PP](2)-InsP(4) were present at 15 pmol/mg (approx. 1.1 microM), 15 pmol/mg (approx. 1.1 microM) and 30 pmol/mg (approx. 2.2 microM) respectively, while the intracellular concentration of InsP(6) was 0.5 nmol/mg of protein (approx. 36 microM). Disruption of aps1 resulted in a 52% decrease in Ap(6)A hydrolase activity in vitro, no detectable change in the intracellular Ap(5)A concentration, and 3-fold increased intracellular concentrations of PP-Ins P(5) and [PP](x)-InsP(x). Disruption of aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30 degrees C. Overexpression of aps1 via two different plasmids that resulted in 60% and 6-fold increases above wild-type enzymic activity in vitro caused no detectable changes in the intracellular concentrations of [PP](2)-InsP(4), [PP](x)-InsP(x) or PP-InsP(5), but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Ap(5)A concentration. Overexpression of aps1 also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multiseptate cells. No phenotypic changes or changes in intracellular Ap(5)A occurred upon overexpression of aps1 E93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades PP-InsP(5) and [PP](x)-InsP(x) in vivo.

The fission yeast Schizosaccharomyces pombe contains a gene on chromosome I that encodes a hypothetical nudix hydrolase, YA9E. The gene, designated aps1, has been cloned and the protein has been purified from Escherichia coli with a yield of 10 mg of Aps1/L of culture. Aps1, composed of 210 amino acids with a calculated molecular mass of 23 724 Da, behaves as a monomer with a sedimentation coefficient of 1.92 S as determined by analytical ultracentrifugation. The effective hydrodynamic radius is about 29 A as determined by both analytical ultracentrifugation and gel-filtration chromatography. Aps1, whose expression was detected in S. pombe by Western blotting, is an enzyme that catalyzes the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being the preferred substrates. The major reaction products are ADP and p4A from Ap6A and ADP and ATP from Ap5A. Values of Km for Ap6A and Ap5A are 19 microM and 22 microM, respectively, and the corresponding values of kcat are 2.0 s-1 and 1.7 s-1, respectively. The enzyme has limited activity on Ap4A and negligible activity on Ap3A, ADP-ribose, and NADH. Aps1 catalyzes the hydrolysis of mononucleotides with decreasing activity in order from p5A to AMP. Optimal activity with Ap6A as substrate is observed at pH 7.6 and in the presence of 0.1-1 mM MnCl2. Aps1 is the first nudix hydrolase isolated from S. pombe, and it is the first enzyme identified with this specific substrate specificity and reaction products.