The influence of inorganic phosphate on the activity of adenosine kinase

The enzyme adenosine kinase (AK; EC 2.7.1.20) shows a dependence upon inorg anic phosphate (Pi) for activity. The degree of dependence varies among enz yme sources and the pH at which the activity is measured. At physiological pH, recombinant AK from Chinese hamster ovary (CHO) cells and AK from beef liver (BL) show higher affinities for the substrate adenosine (Ado), larger maximum velocities and lower sensitivities to substrate inhibition in the presence of Pi. At pH 6.2, both BL and CHO AK exhibit almost complete depen dence on the presence of Pi for activity. The data show that both enzymes e xhibit increasing relief from substrate inhibition upon increasing Pi and t he inhibition of BL AK is almost completely alleviated by the addition of 5 0 mM Pi. The affinity of CHO AK for Ado increases asymptotically from K-m 6 .4 mu M to a limit of 0.7 mu M upon the addition of increasing Pi from 1 to 50 mM. The concentration of Ado necessary to invoke substrate inhibition a lso increases asymptotically from K-i 32 mu M to a limit of 69 mu M at satu rating concentrations of phosphate. In the presence of increasing amounts o f Pi the maximal velocity of activity increases hyperbolically. The effect that phosphate exerts on AK may be either to protect the enzyme from inacti vation at high adenosine and H+ concentrations or to stabilize substrate bi nding at the active site. (C) 2000 Elsevier Science B.V. All rights reserve d.