Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases Potential relationship with the effect of bisphosphonates on osteoclasts.
Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6+/-1.4 mU/mg or 100%; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V(max), K(m), K(cat) and K(cat)/K(m) values were also determined. The K(cat)/K(m) values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl- or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells.
Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols, UAM/CSIC, Facultad de Medicina, 28029 Madrid, Spain. antonio.sillero@uam.es
Maria A Günther
MA
Sillero
Anabel
A
de Diego
Janeth E F
JE
Tavares
Joana A D Catanho da
JA
Silva
Francisco J
FJ
Pérez-Zúñiga
Antonio
A
Sillero
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D011088:DNA Ligases / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D004164:Diphosphonates / Q000494:pharmacology*; D004224:Diterpenes / Q000378:metabolism; D045782:Hemiterpenes / Q000378:metabolism; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism; D013379:Substrate Specificity
D011756:Diphosphates; D004164:Diphosphonates; D004224:Diterpenes; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D011122:Polyphosphates; D012717:Sesquiterpenes; C511282:geranyl diphosphate; C004809:isopentenyl pyrophosphate; C004808:farnesyl pyrophosphate; D000255:Adenosine Triphosphate; D011088:DNA Ligases; D011130:RNA Ligase (ATP); C005692:triphosphoric acid; D008798:Mevalonic Acid