Regulation of smooth-muscle myosin-light-chain kinase. Steady-state kinetic studies of the reaction mechanism

The kinetic mechanism of turkey gizzard smooth muscle myosin-light-chain kinase was investigated using the isolated 20-kDa light chain of myosin as substrate. The kinetic and product inhibition patterns of the forward reaction indicated an ordered sequential mechanism in which MgATP bound first, ADP was released last. The order of substrate binding and product release was confirmed independently by competitive, dead-end inhibition patterns obtained using the non-hydrolizable ATP analog adenosine 5'-beta,gamma-imido]triphosphate. The mechanism was also characterized by a relatively strong product inhibition by ADP and a weak one by phosphorylated 20-kDa light-chain myosin, in addition to a significant inhibition by the latter product via a formation of a dead-end complex. gamma-32P]ATP in equilibrium with 32P]phosphorylated light chain isotope-exchange data were consistent with the deduced mechanism and with the presence of the latter dead-end complex.