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 inorganic phosphate (Pi) for activity. The degree of dependence varies among enzyme 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 dependence on the presence of Pi for activity. The data show that both enzymes exhibit increasing relief from substrate inhibition upon increasing Pi and the inhibition of BL AK is almost completely alleviated by the addition of 50 mM Pi. The affinity of CHO AK for Ado increases asymptotically from K(m) 6.4 microM to a limit of 0.7 microM upon the addition of increasing Pi from 1 to 50 mM. The concentration of Ado necessary to invoke substrate inhibition also increases asymptotically from K(i) 32 microM to a limit of 69 microM at saturating concentrations of phosphate. In the presence of increasing amounts of Pi, the maximal velocity of activity increases hyperbolically. The effect that phosphate exerts on AK may be either to protect the enzyme from inactivation at high adenosine and H(+) concentrations or to stabilize substrate binding at the active site.
Maj
M
M
Department of Biochemistry, McMaster University, Hamilton, Ont., Canada. majm@fhs.mcmaster.ca
Singh
B
B
Gupta
R S
RS
D000241:Adenosine / Q000378:metabolism*; D000248:Adenosine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002942:Circular Dichroism; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010710:Phosphates / Q000494:pharmacology*
D018076:DNA, Complementary; D010710:Phosphates; D000248:Adenosine Kinase; D000241:Adenosine