Kinetics of human soluble and membrane-bound catechol O-methyltransferase: a revised mechanism and description of the thermolabile variant of the enzyme.
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 coli 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 (Vmax) of S-COMT was somewhat higher than that of MB-COMT for all four substrates. The Km values varied considerably, depending on both substrate and enzyme form. S-COMT showed about 15 times higher Km 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 Ks were 3.4 and 20.2 microM 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 [Lundström 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].(ABSTRACT TRUNCATED AT 250 WORDS)
Lotta
T
T
Orion Research, Orion-Farmos, Orion Corporation, Espoo, Finland.
Vidgren
J
J
Tilgmann
C
C
Ulmanen
I
I
Melén
K
K
Julkunen
I
I
Taskinen
J
J
D000818:Animals; D001483:Base Sequence; D002394:Catechol O-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004298:Dopamine / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism
D062385:Hydroxybenzoates; D011994:Recombinant Proteins; C009091:protocatechuic acid; D007980:Levodopa; D012436:S-Adenosylmethionine; D002394:Catechol O-Methyltransferase; D008274:Magnesium; D004298:Dopamine; D009638:Norepinephrine