Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5'-tetraphosphate (p₄A) and adenosine 5'-pentaphosphate (p₅A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5'-O-[-thiotriphosphate] (ATPS), adenosine(5')tetraphospho(5')adenosine (Ap₄A), and adenosine(5')pentaphospho(5')adenosine (Ap₅A) are also substrates of the reaction yielding p₄(d)A in the presence of tripolyphosphate (P₃). UTP, CTP, and AMP are not substrates of the reaction. The K_m values for ATP and P₃ are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl₂ or CoCl₂ equimolecular with the sum of ATP and P₃. The relative rates of synthesis of p₄A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p₄A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p₄A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap₄A (from ATP), Ap₅A (from p₄A), and adenosine(5')tetraphospho(5')nucleoside (Ap₄N) from adequate adenylyl donors (ATP, ATPS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates).