1-(2-AMINOETHYL)-1,4-CYCLOHEXADIENE - A PROBE TO EXAMINE THE CHEMISTRY AND ENERGETICS OF THE C-H BOND-CLEAVAGE IN DOPAMINE BETA-MONOOXYGENASE CATALYSIS

The chemistry and energetics of the initial C-H bond cleavage step in dopamine beta-monooxygenase (D beta M, E.C. 1.14.17.1) catalysis has b een explored using 1-(2-aminoethyl)-1,4-cyclohexadiene (CHDEA) as a pr obe. D beta M has been previously shown (Wimalasena, K.; May, S. W. J. Am. Chem. Soc. 1989, 111, 2729) to catalyze the aromatization of CHDE A to phenylethylamine (PEA). We now report that the side chain hydroxy lated product, 2-amino-1-(1,4-cyclohexadiene)ethanol (CHDEA-OH), is al so a direct product of the D beta M/CHDEA reaction. The PEA:CHDEA-OH p roduct ratio is 2.7 at pH 5.2 and 37 degrees C for the D beta M/CHDEA reaction. The side chain deuterated analog, 1-(2-amino-1,1-dideuterioe thyl)-1,4-cylohexadiene, undergoes aromatization almost exclusively. T he ring deuterated analog, noethyl)-3,3,6,6-tetradeuterio-1,4-cyclohex adiene, favors side chain hydroxylation 2.6 times over aromatization. The hexadeuterio derivative, erioethyl)3,3,6,6-tetradeuterio-1,4-cyclo hexadiene (CHDEA-d(6)), prefers aromatization 3.9 times. The apparent kinetic isotope effects of CHDEA-d(6) are small, 1.8 for k(cat) and 1. 3 for k(cat)/K-m, suggesting that the initial C-H bond cleavage is onl y partly rate limiting in the CHDEA/D beta M reaction analogous to the normal D beta M hydroxylation reaction. The intrinsic isotope effects for the exocyclic and ring methylene hydrogens are estimated to be 9. 9 and 7.0, respectively. The alteration of the product ratio due to de uterium substitution suggests that activation energies of the initial C-H bond cleavage steps for the two pathways are similar. In contrast to the high thermodynamic driving force for the ring methylene hydroge n abstraction, the similar activation energies must be a consequence o f the proximity and/or the relative orientation of the corresponding h ydrogens of the enzyme-bound CHDEA with respect to the activated coppe r oxygen species.