Lysyl-tRNA synthetase, dissociated from the multienzyme complexes of aminoacyl-tRNA synthetases from rat liver, was previously found to be 6-fold more active than the synthetase complex in the enzymatic synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate. The bi-substrate and product inhibition kinetics of the reaction are analyzed. Free lysyl-tRNA synthetase exhibits distinctly different kinetic patterns from those of an 18 S synthetase complex containing lysyl-tRNA synthetase. The 18 S synthetase complex shows kinetic patterns which are consistent with an ordered Bi Uni Uni Bi ping-pong mechanism. Free lysyl-tRNA synthetase shows kinetic patterns consistent with a random mechanism. The differences in the enzymatic properties are attributed to the organization of the supramolecular structure of the synthetase complex. The results suggest that association of the synthetases may affect the mechanisms of the synthesis of AppppA.