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

Lotta, T.; Vidgren, J.; Tilgmann, C. LU ; Ulmanen, I.; Melen, K.; Julkunen, I. and Taskinen, J. (1995) In Biochemistry 34(13). p.4202-4210
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... (More)

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.

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publishing date
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publication status
published
in
Biochemistry
volume
34
issue
13
pages
9 pages
publisher
The American Chemical Society
external identifiers
  • Scopus:0028918413
ISSN
0006-2960
DOI
10.1021/bi00013a008
language
English
LU publication?
no
id
4d15e24b-6043-4e66-b23c-9f3d27099081
date added to LUP
2016-04-11 13:21:07
date last changed
2016-06-29 09:03:35
@misc{4d15e24b-6043-4e66-b23c-9f3d27099081,
  abstract     = {<p>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.</p>},
  author       = {Lotta, T. and Vidgren, J. and Tilgmann, C. and Ulmanen, I. and Melen, K. and Julkunen, I. and Taskinen, J.},
  issn         = {0006-2960},
  language     = {eng},
  number       = {13},
  pages        = {4202--4210},
  publisher    = {ARRAY(0x88fcf90)},
  series       = {Biochemistry},
  title        = {Kinetics of human soluble and membrane-bound catechol O- methyltransferase : A revised mechanism and description of the thermolabile variant of the enzyme},
  url          = {http://dx.doi.org/10.1021/bi00013a008},
  volume       = {34},
  year         = {1995},
}