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 phosphorylase kinase, which contains an intrinsic calmodulin subunit, has been characterized in detail. The inhibition was competitive with respect to phosphorylase b for both the phosphorylase kinase holoenzyme and its isolated catalytic gamma-subunit (minus calmodulin), and the ratios of the Km for phosphorylase to the Ki for melittin were similar for both forms of the kinase. These findings indicate that inhibition of the phosphorylase kinase holoenzyme by melittin is caused predominantly by its interaction with the catalytic subunit of the enzyme, and not with the endogenous calmodulin subunit. Further proof that melittin interacts directly with the catalytic site was obtained when it was observed that melittin was also a substrate for phosphorylase kinase, with a Km that was less than that for phosphorylase b, although the kcat/Km specificity constant was only 1/200th of that for phosphorylase. The apparent tight binding of melittin to the kinase active site could not be readily rationalized by conventional comparison of sequence similarity between melittin and phosphorylase; however, considerable sequence similarity, centered around the convertible seryl residue 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 Ca(2+)-dependent kinase with a calmodulin-binding peptide is discussed.
Paudel
H K
HK
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163.
Xu
Y H
YH
Jarrett
H W
HW
Carlson
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008555:Melitten / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D010763:Phosphorylase b / Q000037:antagonists & inhibitors*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid
D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D008555:Melitten; D010763:Phosphorylase b; D010764:Phosphorylase Kinase