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.

No abstract

Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcythiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.

The synthesis of P1,P4‐bis(5'‐adenosyl)tetraphosphate (Ap4A) has been considered, for a long time, to be catalyzed mainly by some aminoacyl‐tRNA synthetases [Brevet et al. (1989) Proc. Natl. Acad. Sci. USA 86, 8275‐8279]. Recently, yeast Ap4A phosphorylase, acting in reverse (Guranowski et al. (1988) Biochemistry 27, 2959‐2964), was shown to synthesize Ap4A, too. In the case of the synthetases, the intermediate complex E‐aminoacyl‐AMP may serve as donor of AMP to ATP, yielding Ap4A. Here we demonstrate that firefly luciferase (EC 1.13.12.7) which forms the E‐luciferin‐AMP intermediate also synthesizes Ap4A as well as other dinucleoside polyphosphates. We suggest moreover that: other enzymes (mainly synthetases and some transferases), which catalyze the transfer of a nucleotidyl moiety, via nucleotidyl‐containing intermediates and releasing PPi may produce dinucleoside polyphosphates.

Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA-amido conjugates, the most important of which appears to be JA-Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono-and dinucleoside polyphosphates, which are side-reaction products of many enzymes forming acyl $ adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile-conjugates was observed for (±)-JA and 9,10-dihydro-JA, while the rate of conjugation with 12-hydroxy-JA and OPC-4 (3-oxo-2-(2Z-pentenyl)cyclopentane-1-butyric acid) was only about 1-2% that for (±)-JA. Of the two stereoisomers of JA, (À)-JA and (+)-JA, rate of synthesis of the former was about 100-fold faster than for (+)-JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg 2+ , (À)-JA and tripolyphosphate the ligase produces adenosine 5 0 -tetraphosphate (p 4 A); (2) addition of isoleucine to that mixture halts the p 4 A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap 3 A) nor diadenosine tetraphosphate (Ap 4 A) and (4) Ap 4 A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA-Ile.