Characterization of a novel human catechol-O-methyl-transferase mutant with triplet point mutations.
Human catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the transfer of the methyl group to a variety of endogenous and exogenous catechol substrates using S-adenosyl-L-methionine as the methyl donor. This enzymatic O-methylation plays an important role in the inactivation of biologically-active and toxic catechols. A number of studies in recent years have sought to characterize the polymorphism of human COMT, and also to determine the catalytic activity of polymorphic enzymes. We report here the identification of a new haplotype of the human COMT gene with triplet point mutations, which encodes the D51G/S60F/K162R mutant of the soluble COMT and the D101G/S110F/K212R mutant of the membrane-bound COMT. Kinetic analysis showed that these new COMT variants had essentially the same kinetic characteristics and catalytic activity as the wild-type COMTs for the O-methylation of 2-hydroxyestradiol and 4-hydroxyestradiol in vitro, but they have asignificantly reduced thermostability at 37 degrees C. In addition, the mutant enzymes have different binding affinities for S-adenosyl-L-methionine compared with the wild-type COMTs. In agreement with our biochemical observations, molecular modeling studies also showed that the variant human COMT proteins shared nearly the same overall structures as the wild-type proteins. The binding energy values of the mutant COMTs in complex with catechol estrogen substrates were similar to those of the wild-type COMTs bound with the same substrates.
Bai
Hyoung-Woo
HW
Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Wang
Pan
P
Zhu
Bao Ting
BT
D002394:Catechol O-Methyltransferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D002393:Estrogens, Catechol / Q000378:metabolism; D006239:Haplotypes; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D011110:Polymorphism, Genetic; D055550:Protein Stability; D016415:Sequence Alignment
D002393:Estrogens, Catechol; D004958:Estradiol; C001390:2-hydroxyestradiol; C014036:4-hydroxyestradiol; D002394:Catechol O-Methyltransferase