Biopolym. Cell. 1986; 2(1):12-19.
Structure and Function of Biopolymers
Study of ATP and pyrophosphate-binding sites of tryptophanyl-trna synthetase by phosphonate analogs of ATP and pyrophosphate
1Merkulova T. I., 1Nurbekov M. K., 1Kovaleva G. K.
  1. Institute of Molecular Biology, Academy of Sciences of the USSR
    Moscow, USSR


ATP and pyrophosphate-binding sites of bovine tryptophanyl-tRNA-synthetase were studied by phosphonate analogs of ATP and inorganic pyrophosphate — adenosyl-5'-phospho-ryldiphosphonate (Appcp), adenosyl-5'-diphosphonylphosphate (Apcpp) and methylene-diphosphonate (PcP). Methylenediphosphonate can fulfil the role of PP; in the reaction of tryptophanyl adenylate pyrophosphorolysis with the formation of Appcp, the only product of the reaction. Phosphonate analogs of ATP inhibit the reaction of tryptophan-dependent ATP-[32P]PPi- exchange competitively with respect both to ATP (Ki~0.7 mM for Apcpp and ~3.6 mM for Appcp) and PP; (Ki~2.5 mM for both inhibitors). The two analogs interact with the isolated tryptophanyl adenylate-enzyme complex, corresponding phosphonate analogs of Ap4A being formed. On the basis of the results obtained an assumption is made that the observed earlier enzyme pyrophosphorylation by pyrophosphate, which is cleft from ATP in the course of adenylate formation, may be one of the factors preventing the synthesis of Ap4A on tryptophanyl-tRNA synthetase.


[1] Kisselev LL, Favorova OO, Lavrik OI. Biosynthesis of proteins from amino acids to aminoacyl-tRNA. Moscow, Nauka, 1984; 408 p.
[2] Zamecnik P. Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A): its role in cellular metabolism. Anal Biochem. 1983;134(1):1-10.
[3] Blanquet S, Plateau P, Brevet A. The role of zinc in 5',5'-diadenosine tetraphosphate production by aminoacyl-transfer RNA synthetases. Mol Cell Biochem. 1983;52(1):3-11.
[4] Goerlich O, Foeckler R, Holler E. Mechanism of synthesis of adenosine(5')tetraphospho(5')adenosine (AppppA) by aminoacyl-tRNA synthetases. Eur J Biochem. 1982;126(1):135-42.
[5] Kovaleva GK, Holmuratov EG, Nurbekov MK et al. Tryptophanyl-tRNA synthetase and pyrophosphate. Ric sci. 1984; 113:125-132.
[6] Kovaleva G. K., Nurbekov M. K., Merkulova T. I., Kisselev L. L. Preparation and properties of the pyrophosphorylated form of tryptophanyl-tRNA synthetase. Russian Journal of Bioorganic Chemistry 1983, 9 (7):947-953.
[7] Kovaleva GK, Holmuratov EG, Kisselev LL. Tryptophanyl-tRNA synthetase: pyrophosphorylation of the enzyme in the course of adenylate formation? FEBS Lett. 1983;151(1):79-82.
[8] Tarussova N. B., Zavgorodny C. G., Osipova T. I. Organophosphorous analogues of biologically active compounds. XIV. The synthesis of P1P4-bis(5'-adenosyl)tetraphosphate and 5'-nucleosidetriphosphate phosphonate analogues. Russian Journal of Bioorganic Chemistry 1985, 11 (6):802-807.
[9] Tarussova NB, Osipova TI, Biriukov AI et al.. Phosphonate analogs of nucleotide-5'-triphosphates and P1, P4-diadenosine tetraphosphate. Nucl Acids Res. Symp. Ser. 1984. 14:287-288.
[10] Roy KL, Soll D. Fractionation of Escherichia coli transfer RNA on benzoylated DEAE-cellulose. Biochim Biophys Acta. 1968;161(2):572-4.
[11] Kisselev LL, Favorova OO, Kovaleva GK. Tryptophanyl-tRNA synthetase from beef pancreas. Methods Enzymol. 1979;59:234-57.
[12] Fersht AR, Ashford JS, Bruton CJ, Jakes R, Koch GL, Hartley BS. Active site titration and aminoacyl adenylate binding stoichiometry of aminoacyl-tRNA synthetases. Biochemistry. 1975;14(1):1-4.
[13] Freist W, von der Haar F, Cramer F. Isoleucyl-tRNA synthetase from Baker's yeast. Action of ATP analogs in pyrophosphate exchange and aminoacylation, two pathways of the aminoacylation depending on concentration of pyrophosphate. Eur J Biochem. 1981;119(1):151-64.
[14] Freist W, Sternbach H, Cramer F. Arginyl-tRNA synthetase from Baker's yeast. Order of substrate addition and action of ATP analogs in the aminoacylation reaction; influence of pyrophosphate on the catalytic mechanism. Eur J Biochem. 1981;119(3):477-82.
[15] Yount RG. ATP analogs. Adv Enzymol Relat Areas Mol Biol. 1975;43:1-56.
[16] Favorova OO, Kovaleva GK, Moroz SG, Kisselev LL. Tryptophanyl tRNA synthetase: isolation and characteristics of the tryptophanyl-enzyme. Mol Biol (Mosk). 1978;12(3):588-601.
[17] Freist W., Wlender H., Cramer F. Chemically modified ATP derivatives for the study of aminoacyl-tRNA synthetases from baker's yeast: ATP analogs with fixed conformations or modified triphosphate chains in the aminoacylation reaction. Bioorg Chem. 1980; 9(4):491-504.
[18] Marutzky R, Flossdorf J, Kula MR. ATP-analogues as substrates for the leucyl-tRNA synthetase from Escherichia coli MRE 600. Nucleic Acids Res. 1976;3(8):2067-78.
[19] Led JJ, Switon WK, Jensen KF. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography. Eur J Biochem. 1983;136(3):469-79.
[20] Kovaleva GK, Degtiarev SKh, Favorova OO. Affinity modification of tryptophanyl-tRNA synthetase by an alkylating L-tryptophan analog. Mol Biol (Mosk). 1979;13(6):1237-46.