Secondary isotope effects and structure-reactivity correlations in the dopamine .beta.-monooxygenase reaction: evidence for a chemical mechanism

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Abstract
The chemical mechanism of hydroxylation, catalyzed by dopamine .beta.-monooxygenase, was explored with a combination of secondary kinetic isotope effects and structure-reactivity correlations. Measurement of primary and secondary isotope effects on Vmax/Km under conditions where the intrinsic primary hydrogen isotope effect is known allows calculation of the corresponding intrinsic secondary isotope effect. By this method an .alpha.-deuterium isotope effect was obtained, Dk.alpha. = 1.19 .+-. 0.06, with dopamine as substrate. The .beta.-deuterium isotope effect is indistinguishable from one. The large magnitude of Dk.alpha., together with a previous determination of a near maximal primary deuterium isotope effect of 9.4-11, clearly indicates the occurrence of a stepwise process for C-H bond cleavage and C-O bond formation and hence the presence of a substrate-derived intermediate. To probe the nature of this intermediate, a structure-reactivity study was performed by using a series of para-substituted phenylethylamines. Deuterium isotope effects on Vmax and Vmax/Km parameters were determined for all of the substrates, allowing calculation of the rate constants for C-H bond cleavage and product dissociation and dissociation constants for amine and O2 loss from the enzyme-substrate ternary complex. Multiple regression analysis yielded an electronic effect of .rho. = -1.5 for the C-H bond cleavage step, eliminating the possibility of a carbanion intermediate. A negative .rho. value is consistent with formation of either a radical or a carbocation. However, a significantly better correlation is obtained with .sigma.p rather than .sigma.p+, implying formation of a radical intermediate via a polarized transition state. Additional effects determined from the regression analyses include steric effects on rate constants for substrate hydroxylation and product release and on Kdamines, consistent with a sterically restricted binding site, and a positive electronic effect of .rho. = 1.4 on product dissociation, ascribed to a loss of product from an enzyme-bound Cu(II)-alkoxide complex. A mechanism was proposed in which O-O homolysis [from a putative Cu(II)-OOH species] and C-H homolysis (from substrate) occur in a concerted fashion, circumventing the formation of a discrete, high energy O2 species such as hydroxyl radical. The substrate and peroxide-derived radical intermediates thus formed undergo a recombination, kinetically limited by displacement of an intervening water molecule, to give the postulated Cu(II)-alkoxide product complex.