Human soluble (S) and membrane-bound (MB) catechol O-methyltransferase (COMT, EC 184.108.40.206)
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)