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Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase

AuthorsGünther Sillero, María A.; Guranowski, Andrzej; Sillero, Antonio CSIC
Issue DateDec-1991
PublisherBlackwell Publishing
CitationThe Febs Journal 202(2): 507-513 (1993)
AbstractIn the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1, P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2–3 μM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12–20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates: diadenosine 5',5"'-P1, P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1, P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1, P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide {p4A, dATP, adenosine 5'-[α,β-methylene]-triphosphate, (Ap[CH2]pp), (S)-adenosine-5'-[α-thio]triphosphate ((Sp)ATP[αS]) and GTP}, luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1, P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1, P4-[α,β-methylene] tetraphosphate (Ap[CH2]ppA), (Sp-diadenosine 5',5"'-P1, P4-[α-thio]tetraphosphate ((Sp)Ap4A[αS]) and adenosine-5',5"'-P1, P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact α-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1, P3-triphosphate (Ap3A) or adenosine-5',5"'-P1, P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.
Description7 pages, 5 figures, 2 tables.
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Appears in Collections:(IIBM) Artículos

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