title: Kinetic and pH studies on human phenylethanolamine N-methyltransferase.
abstract: Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline) while, concomitantly, S-adenosyl-L-methionine (AdoMet) is converted to S-adenosyl-L-homocysteine. This reaction represents the terminal step in catecholamine biosynthesis and inhibitors of PNMT have been investigated, inter alia, as potential antihypertensive agents. At various times the kinetic mechanism of PNMT has been reported to operate by a random mechanism, an ordered mechanism in which norepinephrine binds first, and an ordered mechanism in which AdoMet binds first. Here we report the results of initial velocity studies on human PNMT in the absence and presence of product and dead end inhibitors. These, coupled with isothermal titration calorimetry and fluorescence binding experiments, clearly shown that hPNMT operates by an ordered sequential mechanism in which AdoMet binds first. Although the logV pH-profile was not well defined, plots of logV/K versus pH for AdoMet and phenylethanolamine, as well as the pKi versus pH for the inhibitor, SK&F 29661, were all bell-shaped indicating that a protonated and an unprotonated group are required for catalysis.
authors.affiliation: Department of Medicinal Chemistry, University of Michigan, 428 Church St, Ann Arbor, MI 48105, United States.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McLeish
mesh_terms: D055162:Biocatalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D010625:Phenylethanolamine N-Methyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D004791:Enzyme Inhibitors; D007546:Isoquinolines; C018428:1,2,3,4-tetrahydroisoquinoline-7-sulfonamide; D010625:Phenylethanolamine N-Methyltransferase
keywords: AdoMet; Adrenaline; Epinephrine; Inhibition; Mechanism; Ordered sequential
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