Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase |
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Authors: | Sillero María A Günther de Diego Anabel Silles Eduardo Pérez-Zúñiga Francisco Sillero Antonio |
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Affiliation: | Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols UAM/CSIC, Facultad de Medicina, 28029 Madrid, Spain. |
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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. |
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Keywords: | Aa, amino acid Ap4A, diadenosine 5′,5?-P1,P4-tetraphosphate Ap4G, adenosine 5′,5?-P1,P4-tetraphosphoguanosine AppCCl2p, adenosine 5′-(β,γ-dichloromethylenetriphosphate) AppCH2p, adenosine 5′-(β,γ-methylenetriphosphate) AppC(R1)(R2)p, adenosine 5′-triphosphate derivative of any bisphosphonate AppC(R1)(R2)ppA, diadenosine 5′,5?-P1,P4-tetraphosphate derivative of any bisphosphonate P3, tripolyphosphate pC(OH)(CH2-CH2-NH2)p, pamidronate or 3-amino-1-hydroxpropylidene-1,1-bisphosphonate pC(OH)(CH2-CH2-CH2-NH2)p, alendronate or 4-amino-1-hydroxybutylidene-1,1-bisphosphonate pC(OH)(CH3)p (HEBP), etidronate or ethane-1-hydroxy-1,1-bisphosphonate or hydroxyethylidenebisphosphonate pCCl2p (Cl2MBP), clodronate or methane-1-dichloro-1,1-bisphosphonate or dichloromethylenebisphosphonate pCH2p (MBP) or methylenebisphosphonate pC(R1)(R2)p, any bisphosphonate |
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