Interaction of non-conjugated olefinic substrate analogues with dopamine beta-monooxygenase: catalysis and mechanism-based inhibition.
The reaction of dopamine beta-monooxygenase (DBM; EC 1.14.17.1) with the prototypical non-conjugated olefinic substrate, 2-(1-cyclohexenyl)ethylamine (CyHEA) [see Sirimanne and May (1988) J. Am. Chem. Soc. 110, 7560-7561], was characterized. CyHEA undergoes facile DBM-catalysed allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic rearrangement, and with stereochemistry consistent with that of DBM-catalysed benzylic hydroxylation and sulphoxidation. The kcat. of 90 s-1 for CyHEA oxygenation is about 75% of the kcat. for tyramine, the substrate commonly used in assays of DBM activity. DBM-catalysed oxygenation of CyHEA also results in mechanism-based inactivation of DBM, with the inactivation reaction yielding kinact. = 0.3 min-1 at pH 5.0 and 37 degrees C, and a partition ratio of 16,000. Although both CyHEA turnover and inactivation exhibit normal kinetics, CyHEA processing also results in gradual depletion of copper from DBM; however, mechanism-based irreversible 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 abstraction 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, kcat. for oxygenation of cis-2-hexenylamine, which also produces only allylic alcohol product, is only 14% of that for CyHEA. Similarly, kinact./KI for turnover-dependent inactivation by the acyclic olefin 2-aminomethyl-1-pentene is more than an order of magnitude smaller than that for benzylic olefins. Our results establish that DBM catalyses allylic oxygenation of a number of non-conjugated 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 radical cation formation from a non-conjugated olefinic moiety. The striking contrast between DBM and cytochrome P-450, which carries out both epoxidation and allylic oxidation with non-conjugated olefinic substrates, is probably a reflection of the differences in redox potential of the activated oxygen species operative for these two enzymes.
Sirimanne
S R
SR
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332.
May
S W
SW
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004299:Dopamine beta-Hydroxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004852:Epoxy Compounds / Q000378:metabolism; D005021:Ethylamines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006900:Hydroxylation; D007659:Ketones / Q000378:metabolism; D007700:Kinetics; D013237:Stereoisomerism
D004852:Epoxy Compounds; D005021:Ethylamines; D007659:Ketones; D003300:Copper; D003577:Cytochrome P-450 Enzyme System; D004299:Dopamine beta-Hydroxylase; C092079:cyclohexylethylamine