Selenophosphate synthetase. Enzyme properties and catalytic reaction.

Selenophosphate synthetase, the product of the selD gene, produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide. Isolation of the enzyme and characterization of some of its physical and catalytic properties are described. Magnesium ion and a monovalent cation, K+, NH4+, or Rb+, are required for catalytic activity. Polyphosphates and other common nucleotide triphosphates do not replace ATP as substrate. The stoichiometry of the catalytic reaction (Reaction 1) was established using 31P NMR, anaerobic molecular sieve chromatography, and radiochemical labeling procedures. ATP+selenide+H2O-->selenophosphate+Pi+AMP. In the absence of selenide, ATP is converted completely to AMP and orthophosphate upon prolonged incubation with elevated levels of enzyme. AMP is a competitive inhibitor of ATP, Ki = 170 microM, whereas selenophosphate and orthophosphate are weak inhibitors indicating a multistep reaction. Attempts to obtain direct evidence for a postulated enzyme-pyrophosphate intermediate using several experimental approaches are described. No exchange of [14C]AMP with ATP could be detected after the enzyme was freed of traces of contaminating adenylate kinase by chromatography on phenyl-Sepharose.

Escherichia coli mutant SELD enzymes. The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide.

Synthesis of a labile selenium donor compound, selenophosphate, from selenide and ATP by the Escherichia coli SELD enzyme was reported previously from this laboratory. From the gene sequence, SELD is a 37-kDa protein that contains 7 cysteine residues, 2 of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys-Ile- (Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and Böck, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 73, 543-547). Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least 1 cysteine residue in the protein is essential for enzyme activity. To test the possibility that the Cys17 and/or Cys19 residue might be essential, these were changed to serine residues by site-specific mutagenesis. The biological activities of the wild type and mutant proteins were studied using E. coli MB08 (selD-) transformed with plasmids containing the selD genes. The plasmid containing the Cys17-mutated gene failed to complement MB08, whereas the Cys19-mutated gene was indistinguishable from wild type. The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix, showing that their affinities for ATP were unimpaired. Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys17, but the Cys19 mutation had no effect on the ability of the enzyme to catalyze the reaction. These results indicate that Cys17 has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.

Synthesis of 5-methylaminomethyl-2-selenouridine in tRNAs: 31P NMR studies show the labile selenium donor synthesized by the selD gene product contains selenium bonded to phosphorus.

An enzyme preparation from Salmonella typhimurium catalyzes the conversion of 5-methylaminomethyl-2-thiouridine in tRNAs to 5-methylaminomethyl-2-selenouridine when supplemented with selenide and ATP. Similar preparations from a Salmonella mutant strain carrying a defective selD gene fail to catalyze this selenium substitution reaction. However, supplementation of the deficient enzyme preparation with the purified selD gene product (SELD protein) restored synthesis of seleno-tRNAs. In the absence of the complementary enzyme(s), the SELD protein catalyzes the synthesis of a labile selenium donor compound from selenide and ATP. 31P NMR studies show that among the products of this reaction are AMP and a compound containing selenium bonded to phosphorus. The reaction is completely dependent on the addition of both selenide and magnesium. The dependence of reaction velocity on ATP concentration shows sigmoidal kinetics, whereas dependence on selenide concentration obeys Michaelis-Menten kinetics indicating a Km value of 46 microM for selenide.

Biosynthesis of selenium-modified tRNAs in Methanococcus vannielii.

Selenium-containing nucleosides are natural components of several tRNA species in Methanococcus vannielii. In the present study, the incorporation of selenium from 75SeO3(2-) into these macromolecules was investigated in sonic extracts of M. vannielii. Nucleoside analysis of the 75Se-labeled tRNAs from these in vitro reaction mixtures demonstrated that the selenium was present in 75Se-labeled nucleosides identical to the two naturally occurring 2-selenouridines produced in vivo. Incorporation of selenium into these nucleosides was ATP-dependent and was maximal after 20 min. Addition of O-acetylserine enhanced the activity 2- to 3-fold, implicating a role for selenocysteine in the reaction. Added L-selenocysteine could function as a selenium donor, but the D isomer and DL-selenomethionine were inactive. RPC-5 chromatography of bulk tRNA isolated from M. vannielii grown on 75SeO3(2-) separated five major species of seleno-tRNAs. The amino acid-accepting activity of these tRNAs was investigated.

In vitro incorporation of selenium into tRNAs of Salmonella typhimurium.

Broken-cell preparations of Salmonella typhimurium rapidly incorporated 75Se from 75SeO3(2-) into tRNA by an ATP-dependent process. Selenium incorporation in the presence of 50 microM 75SeO3(2-) (0.8-1 pmol per A260 unit) was enhanced by the selenocysteine precursor, O-acetyl-L-serine (to 3.7 pmol per A260 unit). This increase in incorporation was a function of O-acetyl-L-serine concentration. Neither O-acetyl-L-homoserine nor O-phospho-L-serine stimulated the incorporation of selenium into tRNA. The incorporation of 75Se from 75SeO3(2-) was decreased by adding L-selenocysteine but not by adding the D isomer. When homologous bulk tRNA was added to the broken-cell preparations, an increased rate of 75Se labeling was observed. The supernatant fraction of the broken-cell preparation contained all of the enzymes required for this process. Reversed-phase HPLC analysis of labeled bulk tRNA digested to nucleosides showed the presence of a labeled compound that coeluted with authentic 5-methylaminomethyl-2-selenouridine.

Biological activity of the potent uridine phosphorylase inhibitor 5-ethyl-2,2'-anhydrouridine.

5-Ethyl-2,2'-anhydrouridine (ANEUR) proved to be a potent inhibitor of uridine phosphorylase isolated from sarcoma 180 cells with an apparent Ki (Ki(app) value of 99 nM. Coadministration of ANEUR with 5-fluorouridine (FUR) resulted in increased toxicity of FUR. The LD50 value of FUR alone was 9 mg/kg (when administered for 5 consecutive days) while the LD50 was 3 mg/kg when FUR was administered together with ANEUR in vivo. There was no significant difference in mean tumor weight on day 10 between control animals and animals treated with FUR (5 mg/kg/day for 3 days) or ANEUR (280 mg/kg/day for 3 days). When FUR was coadministered with ANEUR, mean tumor weight was 91% less than that of the untreated controls, showing that ANEUR, the potent URPase inhibitor, increases the antitumor effect of FUR.