Kinetic studies of rat liver adenosine kinase. Explanation of exchange reaction between adenosine and AMP.

Rat liver adenosine kinase can catalyze an exchange reaction between adenosine and AMP in the absence of ATP (Bontemps, F., Mimouni, M., and Van den Berghe, G. (1993) Biochem. J. 290, 679-684), suggesting a classical ping-pong mechanism. Contrary to expectations, formation of a phosphorylenzyme intermediate could not be demonstrated by incubating the enzyme with [gamma-32P] ATP. Although initial velocity measurements in function of the concentration of adenosine or Mg.ATP, at various fixed concentrations of Mg.ATP or adenosine, generated parallel line patterns, inhibition studies revealed that competitive inhibition was only observed between ADP and ATP. This indicates an Ordered Bi Bi mechanism in which ATP binds first to the enzyme, and ADP is released last. The adenosine-AMP exchange reaction was found to be potently stimulated by ADP, and the basal exchange reaction, i.e. measured in the absence of added ADP, could be accounted for by a slight (0.001%) contamination by ADP of analytical grade AMP. The ADP requirement of the adenosine-AMP exchange reaction explains its occurrence in an Ordered Bi Bi mechanism. Stimulation of the exchange reaction between AMP and adenosine by increasing concentrations of ADP/ATP, and stimulation followed by inhibition of the exchange reaction between ADP and ATP by increasing concentrations of AMP/adenosine, corroborated the proposed mechanism.