Mode of inhibition of smooth muscle myosin light chain kinase by synthetic peptide analogs of the regulatory site.

The functions associated with the inhibitory region and calmodulin binding region of smooth muscle myosin light chain kinase (MLCK) were studied using various synthetic peptide analogs. Peptides 480-501 and 483-498 strongly inhibited 61 kDa Ca2+/calmodulin-independent MLCK activity with Ki of 25 nM. Peptides 493-512 and 493-504 were considerably less effective as inhibitor of the Ca2+/calmodulin-independent MLCK and Kiapp. were 2 and 3 microM, respectively. Inhibition of Ca2+/calmodulin-independent MLCK by the peptides 480-501 and 483-498 were competitive with ATP and 20,000 dalton smooth muscle myosin light chain. The inhibition of native MLCK by peptide 493-512 was explained by the calmodulin depletion model in which the peptide binds to free calmodulin and prevents it from activating MLCK. On the other hand, the inhibition of native MLCK by the peptides 480-501 and 483-498 was explained by the binding of these peptides to the MLCK-calmodulin complex. The present study suggests that the inhibitory region of MLCK directly binds to MLCK active site and competes with both ATP and 20,000 dalton light chain so as to inhibit the enzyme.