THE MODEL CALMODULIN-BINDING PEPTIDE MELITTIN INHIBITS PHOSPHORYLASE-KINASE BY INTERACTING WITH ITS CATALYTIC CENTER

The inhibition by melittin, a model calmodulin-binding peptide, of pho sphorylase kinase, which contains an intrinsic calmodulin subunit, has been characterized in detail. The inhibition was competitive with res pect to phosphorylase b for both the phosphorylase kinase holoenzyme a nd its isolated catalytic gamma-subunit (minus calmodulin), and the ra tios of the K(m) for phosphorylase to the K(i) for melittin were simil ar for both forms of the kinase. These findings indicate that inhibiti on of the phosphorylase kinase holoenzyme by melittin is caused predom inantly by its interaction with the catalytic subunit of the enzyme, a nd not with the endogenous calmodulin subunit. Further proof that meli ttin interacts directly with the catalytic site was obtained when it w as observed that melittin was also a substrate for phosphorylase kinas e, with a K(m) that was less than that for phosphorylase b, although t he k(cat)/K(m) specificity constant was only 1/200th of that for phosp horylase. The apparent tight binding of melittin to the kinase active site could not be readily rationalized by conventional comparison of s equence similarity between melittin and phosphorylase; however, consid erable sequence similarity, centered around the convertible seryl resi due of phosphorylase, was observed when the sequences were aligned in reversed polarity. The possible regulatory significance of the direct interaction of the catalytic site of this Ca2+-dependent kinase with a calmodulin-binding peptide is discussed.