INTERACTION OF NONCONJUGATED OLEFINIC SUBSTRATE-ANALOGS WITH DOPAMINEBETA-MONOOXYGENASE - CATALYSIS AND MECHANISM-BASED INHIBITION

The reaction of dopamine beta-monooxygenase (DBM; EC 1.14.17.1) with t he prototypical non-conjugated olefinic substrate, 2-(1-cyclohexenyl)e thylamine (CyHEA) [see Sirimanne and May (1988) J. Am. Chem. Sec. 110, 756-7561], was characterized. CyHEA undergoes facile DBM-catalysed al lylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyH EA-OH) without detectable epoxidation or allylic rearrangement, and wi th stereochemistry consistent with that of DBM-catalysed benzylic hydr oxylation and sulphoxidation. The k(cat) of 90 s(-1) for CyHEA oxygena tion is about 75 % of the k(cat). for tyramine, the substrate commonly used in assays of DBM activity. DBM-catalysed oxygenation of CyHEA al so results in mechanism-based inactivation of DBM, with the inactivati on reaction yielding k(inact) = 0.3 min(-1) at pH 5.0 and 37 degrees C , and a partition ratio of 16000. Although both CyHEA turnover and ina ctivation exhibit normal kinetics, CyHEA processing also results in gr adual depletion of copper from DBM; however, mechanism-based irreversi ble DBM inactivation occurs independent of this copper depletion when sufficient copper is present in the assay solution. A likely mechanism for turnover-dependent DBM inactivation by CyHEA involves initial abs traction of an allylic hydrogen to form a resonance-stabilized allylic radical, which can then either partition to product or undergo attack by an active-site residue. Acyclic, non-conjugated olefinic analogues exhibit diminished substrate activity toward DBM. Thus, k(cat) for ox ygenation of cis-2-hexenylamine, which also produces only allylic alco hol product, is only 14 % of that for CyHEA. Similarly, k(inact)/K-I, for turnover-dependent inactivation by the acyclic olefin 2-aminomethy l-1-pentene is more than an order of magnitude smaller than that for b enzylic olefins, Our results establish that DBM catalyses allylic oxyg enation of a number of nonconjugated olefinic substrate analogues with neither epoxidation nor allylic rearrangement occurring. The absence of epoxide products from non-conjugated olefinic substrates implies an inability of the activated copper-oxygen species of DBM to effect rad ical cation formation from a non-conjugated olefinic moiety. The strik ing contrast between DBM and cytochrome P-450, which carries out both epoxidation and allylic oxidation with non-conjugated olefinic substra tes, is probably a reflection of the differences in redox potential of the activated oxygen species operative for these two enzymes.