Kinetics of human soluble and membrane-bound catechol O- methyltransferase: A revised mechanism and description of the thermolabile variant of the enzyme

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Abstract

Human soluble (S) and membrane-bound (MB) catechol O-methyltransferase (COMT, EC 2.1.1.6) enzymes have been expressed at sufficiently high levels in Escherichia coil and in baculovirus-infected insect cells to allow kinetic characterization of the enzyme forms. The use of tight-binding inhibitors such as entacapone enabled the estimation of actual enzyme concentrations and, thereby, comparison of velocity parameters, substrate selectivity, and regioselectivity of the methylation of both enzyme forms. Kinetics of the methylation reaction of dopamine, (-)-noradrenaline, L-dopa, and 3,4- dihydroxybenzoic acid was studied in detail. Here, the catalytic number (V(max)) of S-COMT was somewhat higher than that of MB-COMT for all four substrates. The K(m) values varied considerably, depending on both substrate and enzyme form. S-COMT showed about 15 times higher K(m) values for catecholamines than MB-COMT. The distinctive difference between the enzyme forms was also the higher affinity of MB-COMT for the coenzyme S-adenosyl-L- methionine (AdoMet). The average dissociation constants K(s) were 3.4 and 20.2 μM for MB-COMT and S-COMT, respectively. Comparison between the kinetic results and the atomic structure of S-COMT is presented, and a revised mechanism for the reaction cycle is discussed. Two recently published human COMT cDNA sequences differed in the position of S-COMT amino acid 108, the residue being either Val-108 [Lundstrom et al. (1991) DNA Cell. Biol. 10, 181-189] or Met-108 [Bertocci et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1416-1420]. The catalytic activities of these two COMT variants, expressed in E. coli, were similar, but the Met-108 enzyme was more thermolabile already at physiological temperature (37 °C). The reported existence of a common polymorphism of the human COMT gene coding for a thermolabile low activity, COMT(L), and a thermostable high activity, COMT(H), is discussed in light of the different thermostability of the two enzyme forms.

Details

Authors
  • T. Lotta
  • J. Vidgren
  • C. Tilgmann
  • I. Ulmanen
  • K. Melen
  • I. Julkunen
  • J. Taskinen
External organisations
  • Orion Corporation
Original languageEnglish
Pages (from-to)4202-4210
Number of pages9
JournalBiochemistry
Volume34
Issue number13
Publication statusPublished - 1995
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes