Kinetic studies of adenosine kinase from L1210 cells: a model enzyme with a two-site ping-pong mechanism

Abstract

Purified adenosine kinase from [mouse leukemia] L1210 cells displayed substrate inhibition by high concentrations of adenosine (Ado), ATP, and MgCl2. When incubated with ATP and MgCl2, the enzyme was phosphorylated, and the phosphorylated kinase transferred phosphate to Ado in the absence of ATP and MgCl2. Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a 2-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among many possible pathways within this mechanism probably a random-bi ordered-bi route is the preferred sequence in which the 2 substrates, Ado and MgATP, bind in a random order to form the ternary complex MgATP.cntdot.E.cntdot.Ado followed by the sequential dissociation of MgADP and AMP. Kd of various enzyme-substrate and enzyme-product complexes and the first-order rate constant of the rate-limiting step were estimated.