The kinetic behaviour of myosin light chain kinase isolated from skeletal muscle was studied under steadystate conditions using highly purified phosphorylatable light chains 2 (LC2).Forward reaction, product inhibition, and reverse reaction data indicate a sequential mechanism which can be interpreted best by a rapid-equilibrium random bi-bi reaction model. The forward reaction parameters are KATp = 150 pM, KLc2 = 5.3 pM, and Ki. L-c2 = 7.6 pM. The enzyme forms a dead-end complex with ADP and light chain 2; K d , ADP of this complex is 50 pM. The forward reaction is also strongly inhibited by the phosphorylated light chain 2, Ki, L c 2~ is 1.5 pM. An equilibrium constant Kcq of about 70 can be calculated from the kinetic parameters which agrees with the directly measured value of about 60.The role of the two inhibitory mechanisms in the regulation of the enzyme and of the high energy of the light chain phosphate bond as deducible from Keq are discussed. [16] and, as suggested very recently, inhibition by phosphorylated myosin [I71 may also play a role in the regulation of MLCK activity. In addition, the phosphorylation of the two heads of myosin may be non-random, but there is no agreement about the precise mechanism [17 -191. Until now, knowledge about this kind of bisubstrate reaction and about the inhibitory functions of products is incomplete; however, it may be helpful to understand the regulation of MLCK action. Therefore we have studied in detail the steady-state kinetics of the skeletal muscle enzyme, including product inhibition patterns, the reverse reaction, and the equilibrium constant.In vivo most or all of LC2 is tightly bound to myosin heavy chain. However the non-physiological substrate, isolated LC2, has several essential advantages over native myosin. It

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