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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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.; Ulmanen, I.; Melen, K.; Julkunen, I.; Taskinen, J.

In: Biochemistry, Vol. 34, No. 13, 1995, p. 4202-4210.

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Lotta, T. ; Vidgren, J. ; Tilgmann, C. ; Ulmanen, I. ; Melen, K. ; Julkunen, I. ; Taskinen, J. / Kinetics of human soluble and membrane-bound catechol O- methyltransferase : A revised mechanism and description of the thermolabile variant of the enzyme. In: Biochemistry. 1995 ; Vol. 34, No. 13. pp. 4202-4210.

RIS

TY - JOUR

T1 - Kinetics of human soluble and membrane-bound catechol O- methyltransferase

T2 - A revised mechanism and description of the thermolabile variant of the enzyme

AU - Lotta, T.

AU - Vidgren, J.

AU - Tilgmann, C.

AU - Ulmanen, I.

AU - Melen, K.

AU - Julkunen, I.

AU - Taskinen, J.

PY - 1995

Y1 - 1995

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0028918413&partnerID=8YFLogxK

U2 - 10.1021/bi00013a008

DO - 10.1021/bi00013a008

M3 - Article

VL - 34

SP - 4202

EP - 4210

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 13

ER -