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Glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis was modified with various chemical modifiers to determine the active sites of the enzyme. Treatment of the enzyme with group-specific reagents diethylpyrocarbonate, N-bromosuccinimide, or carbodiimide resulted in complete loss of enzyme activity, which shows histidine, tryptophan, and glutamic acid or aspartic acid residues are at or near the active site. In each case, inactivation followed pseudo first-order kinetics. Inclusion of glycerol-3-phosphate and/or CTP prevented the inactivation, indicating the presence of tryptophan and glutamic acid or aspartic acid residues at the substrate binding site. Analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a two histidine residues, single tryptophan residue, and two glutamic acid or aspartic acid residues.

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참고문헌 (15)

  1. Chemical modification of chloroperoxidase with diethylpyrocarbonate. Evidence for the presence of an essential histidine residue , Blanke, S. R.;L. P. Hager , J. Biol. Chem. / v.265,pp.12454-12461, 1990
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  4. Cloning and expression of rat liver CTP: phosphocholine cytidylyltransferase: an amphipatic protein that controls phosphatidylcholine synthesis , Kalmer, G. B.;R. J. Kar;A. Lachance;R. Aebersold;R. B. Cornell , Proc. Natl. Acad. Sci. USA / v.87,pp.6029-6033, 1990
  5. Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine AC-N lyase , Takeuchi, M.;N. Asano;Y. Kameda;K. Matsui , J. Biochem. / v.99,pp.1571-1577, 1986
  6. Expression, purification, and characterization of CTP: glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis , Park, Y. S.;T. D. Switzer;J. E. Dixon;C. Kent , J. Biol. Chem. / v.268,pp.16648-16654, 1993
  7. Regulation of the biosynthesis of triacylglycerol, phosphatidylcholine and phosphatidyleth-anolamine in the liver , Tijburg, L. B. M.;M. J. H. Geelen;L. M. G. Van Golde , Biochim. Biophys. Acta / v.1004,pp.1-19, 1989
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  9. CTP: phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro , J. S. Sanghera;D. E. Vance , J. Biol. Chem. / v.264,pp.1215-1223, 1989
  10. Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin , Church, F. C.;R. L. Lundblad;C. M. Noyes , J. Biol. Chem. / v.260,pp.4936-4940, 1985
  11. CTP: phosphorylcholine cytidylyltransferase from rat liver , Feldman, D. A.;P. A. Weinhold , J. Biol. Chem. / v.262,pp.9075-9081, 1987
  12. The reactivity toward N-bromosuccinimide of tryptophan in enzymes, zymogens, and inhibited enzymes , Spande, T. F.;N. M. Green;B. Witkop , Biochem. / v.5,pp.1926-1933, 1966
  13. Genes concernd with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units , Mauel, C.;M. Young;D. Karamata , J. Gen. Microbiol. / v.137,pp.929-941, 1991
  14. Chemical evidence for the involvement of tryptophan in the interaction of trypsin with the inhibitor from beef panceas , Spande, T. F.;B. Witkop , Biochem. Biophys. Res. Commun. / v.21,pp.131-134, 1965
  15. Regulation of phosphatidylcholine biosynthesis , Kent, C. , Prog. Lipid Res. / v.29,pp.87-105, 1990

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