Calmodulin N-methyltransferase. Kinetics, mechanism, and inhibitors.
The present study was undertaken to determine kinetic and inhibition parameters and the mechanism of S-adenosyl-L-methionine:calmodulin-L-lysine N6-methyltransferase (EC 2.1.1.60, CLNMT), an enzyme for which calmodulin is a substrate. Partially purified CLNMT isolated from rat testes had a Vmax of 540 pmol/min/mg and Km values for mushroom demethylcalmodulin and S-adenosyl-L-methionine of 230 nM and 2.0 microM, respectively. Kinetic analysis indicated a complex Bi Bi sequential kinetic mechanism for CLNMT where S-adenosyl-L-methionine binds initially and is followed by demethylcalmodulin binding. When the effects of 20 different compounds that are either inhibitors of calmodulin-specific or methylation-specific functions were examined, CLNMT displayed a pattern of inhibition which differs from that seen with calmodulin-activated enzymes. The product of calmodulin methylation, fully trimethylated calmodulin, and nonmethylatable VU-3 calmodulin acted as competitive inhibitors of CLNMT, with Ki values of 310 and 400 nM, respectively. Of the 13 compounds tested, which are inhibitors of calmodulin-dependent cyclic nucleotide phosphodiesterase, only the calmodulin-binding domain from Ca2+/calmodulin-dependent kinase II, melittin, and calmidazolium were effective inhibitors of CLNMT and each exhibited a complex pattern of inhibition with Kis values of 21, 50, and 65 nM, respectively. The only potent methylation-specific inhibitor was S-adenosyl-L-homocysteine, which also displayed a complex pattern of inhibition.
Wright
L S
LS
Department of Pediatrics, University of Wisconsin Medical School, Madison, Wisconsin 53705, USA.
Bertics
P J
PJ
Siegel
F L
FL
D000818:Animals; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D013379:Substrate Specificity
D002147:Calmodulin; D004791:Enzyme Inhibitors; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C026647:calmodulin methyltransferase