Interaction of non-conjugated olefinic substrate analogues with dopamine β-monooxygenase: catalysis and mechanism-based inhibition

Abstract

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.

Characterization of natural vasostatin‐containing peptides in rat heart

“…Oxidation of CGA residues has previously been described [61]. In chromaffin granules, for instance, MS analysis has revealed oxidation at methionine residues, which may be related to a redox mechanism inside the granules [63].…”

“…In particular, in the isolated working heart of the frog Rana esculenta, the concentration-dependent negative inotropism and inhibition of the b-adrenergicdependent positive inotropy elicited by the N-terminal domain of CGA [i.e. human recombinant vasostatin I and bovine CGA(7-57)], as well as by the synthetic peptides corresponding to frog and bovine CGA(4-16), CGA (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66), and bovine CGA(1- 40), have been analysed in details [32]. More recently, using the Langendorff-perfused rat heart model, we have reported that the two recombinant human vasostatins STA-CGA(1-76) (vasostatin I) and STA-CGA(1-113) (vasostatin II) display similar negative inotropic effects and antagonize the b-adrenergic-dependent positive inotropism, the latter being counteracted by vasostatin I in a noncompetitive type of antagonism [33].…”

Inactivation of peptidylglycineα-hydroxylating monooxygenase by cinnamic acid analogs

“…Other olefinic inactivators of PHM include monoethyl fumarate 25 , styrylthioa-cetate 38 , 2- and 3-alkenoates 32 , vinylglycine 32 , 3-substituted acrylates 32 , substituted 4-oxo-2-hexenoates 30 , and D-Phe-L-Phe-L-vinylglycine 64 . Other monooxygenases, P 450 and dopamine β-monooxygenase (DβM, structurally and mechanistically related to PHM), are inactivated by olefins 65–68 . Most of these olefinic inactivators exhibit characteristics of suicide substrates: the inactivation only occurs during enzyme turnover and substrates or competitive inhibitors protect the monooxygenases against inactivation.…”

“…Copper-free PHM is catalytically inactive 53,62,78 . Cinnamates are known to form complexes with both Cu(I) 79,80 and Cu(II) 81 and copper removal/chelation has been attributed to specific PHM 61 and DβM inhibitors 68 . PHM inactivation requires turnover; thus, inactivation of PHM by cinnamate could result from the formation of a relatively high affinity cinnamate–Cu(I) complex.…”

Foreign Compounds and Intermediary Metabolism: Sulfoxidation Bridges the Divide

“…Further work has revealed that this enzyme is able to exhibit a broad specificity towards its organic substrate. Many other molecules, in addition to dopamine, have been shown to succumb to its catalytic activity [62][63][64][65][66][67][68][69][70][71][72].…”