title: Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase.
abstract: T4 RNA ligase catalyzes the synthesis of ATP beta,gamma-bisphosphonate 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(-1)); clodronate, 0.54+/-0.14 mM (0.29+/-0.05 s(-1)); and etidronate, 4.3+/-0.5 mM (0.028+/-0.013 s(-1)). In the presence of GTP, and ATP or AppCCl(2)p the relative rate of synthesis of adenosine 5',5'''-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 beta,gamma-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.
authors.affiliation: Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols UAM/CSIC, Facultad de Medicina, 28029 Madrid, Spain.
authors.forename: María A Günther
authors.initials: MA
authors.lastname: Sillero
authors.forename: Anabel
authors.initials: A
authors.lastname: de Diego
authors.forename: Eduardo
authors.initials: E
authors.lastname: Silles
authors.forename: Francisco
authors.initials: F
authors.lastname: Pérez-Zúñiga
authors.forename: Antonio
authors.initials: A
authors.lastname: Sillero
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004002:Clodronic Acid / Q000378:metabolism; D004164:Diphosphonates / Q000378:metabolism*; D012968:Etidronic Acid / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004164:Diphosphonates; D004002:Clodronic Acid; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; D011130:RNA Ligase (ATP); D012968:Etidronic Acid