Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase

In 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.