Intrasubstituent isotope effect studies of oxidative N-demethylations catalyzed by secondary amine monooxygenase. Comparison to cytochrome P-450

Article abstract of DOI:10.1021/ja00035a059

Intermolecular, intramolecular, and intrasubstituent kinetic deuterium isotope effect studies have been carried out on secondary amine monooxygenase, the only heme-containing monooxygenase other than cytochrome P-450. Secondary amine monooxygenase exhibits an active site structure similar to myoglobin but has a reactivity, oxidative N-dealkylation, in common with those demonstrated by P-450. An mfermolecular isotope effect was not observed for N-dealkylation of dimethylamine, indicating that C-H bond cleavage is not the rate limiting step. The intramolecular isotope effect is also masked by a high commitment to catalysis and/or the absence of substrate methyl group interchange at the active site. A net intrinsic kinetic deuterium isotope effect of 1.76 has been revealed via an intrasubstituent isotope effect study of competition between D and H atoms within each methyl group of dimethylamine in 1,1,1′,1′-tetradeuteriodimethylamine. This value is essentially identical to that previously reported for P-450 and is much smaller than values observed for other histidine-ligated heme proteins such as myoglobin and the peroxidases (Miwa, G. T. et al. J. Biol. Chem. 1983, 258, 14445-14449). The small isotope effect is most consistent with a deprotonation mechanism in which significant H-atom tunneling is absent, rather than a H-atom abstraction mechanism. It has been shown that the identity of the eventual proton acceptor is important in determining the magnitude of the intrinsic isotope effect for deprotonations (Dinnocenzo, J. P.; Banach, T. E. J. Am. Chem. Soc. 1989, 111, 8646-8653). Therefore, a relatively nonpolar proton acceptor environment within secondary amine monooxygenase, like that of cytochrome P-450, is implicated by close agreement in the isotope effects of these two proteins. Since cytochrome P-450 has a thiolate proximal ligand and secondary amine monooxygenase has a histidine proximal ligand, the identity of the heme proximal ligand appears to be less important than the nature of the distal active site environment in these N-dealkylation reactions. Thus, secondary amine monooxygenase resembles myoglobin and the peroxidases in its heme ligation and spectroscopic properties but catalyzes N-dealkylations via a mechanism that is similar to that employed by the other heme-containing monooxygenase, cytochrome P-450.