It was previously assumed that EWLH P -AMP was the intermediate complex in the synthesis of Ap R A catalyzed by firefly luciferase (EC 1.13.12.7), when luciferin (LH P ) was used as cofactor. However, here we show that LH P is partly transformed, shortly after the onset of the luciferase reaction, to dehydroluciferin (L) with formation of an EWL-AMP complex which is the main intermediate for the synthesis of Ap R A. Formation of three more derivatives of LH P were also observed, related to the production of light by the enzyme. CoA, a known stimulator of light production, inhibits the synthesis of Ap R A by reacting with the EWL-AMP complex and yielding L-CoA.z 1998 Federation of European Biochemical Societies.

T4 RNA ligase catalyzes the synthesis of ATP b,cbisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH 2 p) (100), clodronate (pCCl 2 p) (52), and etidronate (pC(OH)(CH 3 )p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH 2 -CH 2 -NH 2 )p), and alendronate (pC(OH)(CH 2 -CH 2 -CH 2 -NH 2 )p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K m values and the rate of synthesis (k cat ) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26 ± 0.05 mM (0.28 ± 0.05 s À1 ); clodronate, 0.54 ± 0.14 mM (0.29 ± 0.05 s À1 ); and etidronate, 4.3 ± 0.5 mM (0.028 ± 0.013 s À1 ). In the presence of GTP, and ATP or AppCCl 2 p the relative rate of synthesis of adenosine 5 0 ,5 000 -P 1 ,P 4 -tetraphosphoguanosine (Ap 4 G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH 2 p) was a very poor substrate for the synthesis of Ap 4 G. To our knowledge this report describes, for the first time, the synthesis of ATP b,c-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3′‐OH residue of a variety of nucleosides, nucleoside 5′‐phosphates and dinucleotides of the type nucleoside(5′)oligophospho(5′)nucleoside is described here for the first time. Using micromolar concentrations of [α‐32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5′)diphospho(5′)adenosine (Ap2A), adenosine (5′)triphospho(5′)adenosine (Ap3A), adenosine(5′)tetraphospho(5′)adenosine (Ap4A), adenosine(5′)pentaphospho(5′)adenosine (Ap5A), guanosine(5′)diphospho(5′) guanosine (Gp2G), guanosine(5′)triphospho(5′)guanosine (Gp3G), guanosine(5′)tetraphospho(5′)guanosine (Gp4G), and guanosine(5′)pentaphospho(5′)guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase. The presence of 1 mm GMP inhibited competitively the polyadenylylation of tRNA. We hypothesize that the type of methods used to measure polyadenylation of RNA is the reason why this novel property of E. coli poly(A) polymerase has not been observed previously.