Isolation and kinetic characterization of the calmodulin methyltransferase from sheep brain.
The methyltransferase that catalyzes the trimethylation of lysine 115 in calmodulin has been purified from sheep brain. The enzyme is a monomer with an apparent molecular weight of 38,000 on the basis of gel filtration chromatography and SDS-polyacrylamide electrophoresis. In the presence of calcium the methyltransferase exhibited a Km of 100 nM for unmethylated calmodulin and a kcat of 0.0278 s-1. The enzyme was able to use calcium-depleted calmodulin as a substrate, albeit with less efficiency. The methylation of calcium-depleted calmodulin was inhibited by increases in ionic strength, whereas methylation of calcium-saturated calmodulin was not affected. This suggests a difference in the mode of interaction of calcium-saturated and calcium-depleted calmodulins with the enzyme. As with calmodulin's interactions with other calmodulin-dependent enzymes, the oxidation of the methionines of calmodulin by performic acid treatment decreases the ability of the methyltransferase to recognize and methylate calmodulin. A calmodulin-binding peptide based on the calmodulin-dependent protein kinase II sequence and the naphthalenesulfonamide W-7 inhibit the calmodulin methyltransferase-calmodulin interaction in a calcium-dependent manner. Removal of the NH2-terminal lobe (residues 1-77) does not affect the ability of the calmodulin methyltransferase to recognize and methylate lysine 115. Thus, the determinants for calmodulin methyltransferase binding reside solely in the COOH-terminal lobe of calmodulin. Further, structural features within this region, in particular, the hydrophobic cleft, that are manifested upon calcium binding may contribute to the interaction of calmodulin with the enzyme.
Han
C H
CH
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Richardson
J
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Oh
S H
SH
Roberts
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DM
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D012756:Sheep
D002147:Calmodulin; D010455:Peptides; D008715:Methionine; D008780:Methyltransferases; C026647:calmodulin methyltransferase