TAILIEUCHUNG - Báo cáo khoa học: The duality of LysU, a catalyst for both Ap4A and Ap3A formation

Heat shock inducible lysyl-tRNA synthetase ofEscherichia coli(LysU) is known to be a highly efficient diadenosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate (Ap4A) synthase. However, we use an ion-exchange HPLC technique to demonstrate that active LysU mixtures actually have a dual catalytic activ-ity, initially producing Ap4 A from ATP, before converting that tetraphos-phate to a triphosphate. | iFEBS Journal The duality of LysU a catalyst for both Ap4A and Ap3A formation Michael Wright Nonlawat Boonyalai Julian A. Tanner Alison D. Hindley and Andrew D. Miller Imperial college Genetic Therapies Centre Department of Chemistry Imperial college London London UK Keywords Ap4A Ap3A dinucleoside polyphosphates heat shock response LysU Correspondence A. D. Miller ImperialCollege Genetic Therapies Centre Department of Chemistry Flowers Building Armstrong Road Imperial College London London SW7 2AZ UK Fax 44 20 75945803 Tel 44 20 75945773 E-mail Received 21 March 2006 revised 31 May accepted 6 June 2006 doi Heat shock inducible lysyl-tRNA synthetase of Escherichia coli LysU is known to be a highly efficient diadenosine 5 .5 - j1. j4-tetraphosphate Ap4A synthase. However we use an ion-exchange HPLC technique to demonstrate that active LysU mixtures actually have a dual catalytic activity initially producing Ap4A from ATP before converting that tetraphosphate to a triphosphate. LysU appears to be an effective diadenosine 5 .5 - j1. j3-triphosphate Ap3A synthase. Mechanistic investigations reveal that Ap3A formation requires a that the second step of Ap4A formation is slightly reversible thereby leading to a modest reappearance of adenylate intermediate and b that phosphate is present to trap the intermediate either as inorganic phosphate as added ADP or as ADP generated in situ from inorganic phosphate . Ap3A forms readily from Ap4A in the presence of such phosphate-based adenylate traps via a reverse-trap mechanism . LysU is also clearly demonstrated to exist in a phosphorylated state that is more physically robust as a catalyst of Ap4A formation than the nonphosphorylated state. However phosphorylated LysU shows only marginally improved catalytic efficiency. We note that Ap3A effects have barely been studied in prokaryotic organisms. By contrast there is a body of literature that describes Ap3A and Ap4A .

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