Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases.

Abstract T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP β,γ-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH 3 )p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K m values and the rate of synthesis k cat (s −1 ), determined for the following compounds were, respectively: etidronate, 0.73 ± 0.09 mM and (70 ± 10) × 10 −3  s −1 ; clodronate (pCCl 2 p), 0.08 ± 0.01 mM and (4.1 ± 0.3) × 10 −3  s −1 ; methylenebisphosphonate (pCH 2 p), 0.024 ± 0.001 mM and (0.6 ± 0.1) × 10 −3  s −1 ; tripolyphosphate (P 3 ) (in the synthesis of adenosine 5′-tetraphosphate, p 4 A), 1.30 ± 0.30 mM and (6.2 ± 1.1) × 10 −3  s −1 ; (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap 4 G was observed with clodronate but not with pamidronate (pC(OH)(CH 2 –CH 2 –NH 3 )p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative ( K m  = 0.36 ± 0.09 mM and k cat  = 0.15 ± 0.02 s −1 ). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.