Mechanism-based inactivation of porcine kidney diamine oxidase by 1,4-diamino-2-butene.

Cis- and trans-1,4-diamino-2-butene are substrates and potent inactivators of porcine kidney diamine oxidase. Evidence from absorption and NMR spectra indicates that both are oxidized to pyrrole. Both substrates are irreversible mechanism-based inactivators of the enzyme, although the trans isomer is more potent and results in complete inactivation in a reaction which follows pseudo-first-order kinetics with an apparent Ki of 0.34 mM and a second-order inactivation constant of 500 M-1 s-1. Under the same conditions, 46% of the activity remains when the enzyme is reacted with cis-1,4-diamino-2-butene. Trans-4-amino-2-butenal, the product of oxidation of the trans diamine, has been synthesized and shown to undergo cyclization to pyrrole in a concentration-dependent manner, approaching second-order at low concentrations. Trans-4-amino-2-butenal is itself a potent irreversible inhibitor with IC50 of 2.5 microM. We propose that the irreversible inactivation by both cis- and trans-1,4-diamino-2-butene involves attack by a protein-based nucleophilic residue on the unsaturated aminoenal products of the enzymatic reactions, resulting in a covalent adduct. Cyclization of the cis-aminoenal to pyrrole is much more rapid than in the trans case, thus it is less available for inhibitory reaction with the protein.

Haemoprotein-mediated metabolism of enamines and the possible involvement of one-electron oxidations.

1. Microsomal metabolism of 1-benzylpiperidine (1-BP), its cis-2,6-dimethyl (cis-2,6-DMBP), 4,4-dimethyl (4,4-DMBP), and alpha, alpha-dimethyl (alpha, alpha-DMBP) analogues, and phencyclidine (PCP) has been studied to assess the involvement of P450 oxidation of the enamine tautomers of the initial endocyclic iminium metabolites. 2. The selective prevention by cyanide of the metabolite production of 1-benzyl-3-piperidone but not 1-benzyl-3-piperidinol from 1-BP is consistent with the enamine as the source of the 3-one metabolite. 3. The parent amines and particularly the independently prepared iminium species induced a pattern of metabolism-dependent irreversible inactivation of P450 benz-phetamine demethylase activity, consistent with involvement of enamine C-3 oxidation in the inactivation process. 4. Substrate activity of the endocyclic enamines and alpha-aminoketones (presumably the enol-enamine tautomers) for horseradish peroxidase under conditions where simple aliphatic amines display no activity is consistent with metabolic one-electron oxidations of the enamines.

Structural definition of early lysine and histidine adduction chemistry of 4-hydroxynonenal.

The lipid peroxidation product trans-4-hydroxy-2-nonenal (HNE) has been implicated in the covalent modification of low-density lipoproteins (LDL) thought to contribute to the over-accumulation of LDL in the arterial wall in the initial stages of atherosclerosis. Proposals for the exact structures of "early" protein side-chain modifications until now have been based on indirect evidence. In this paper, the structures of first-formed His- and Lys-based adducts were elucidated by correlating NMR spectral properties with those obtained on models with reduced chiral center content, in some cases following hydride reduction. In this manner, we could confirm unambiguously the structure of a HNE-His imidazole(N tau) Michael adduct, stabilized as a cyclic hemiacetal and isolated from a neutral aqueous 1:1 stoichiometry reaction mixture. In the case of Lys/amine reactivity, where an excess of amine is needed to avert HNE aldol condensation, the predominance of a 1:1 Michael adduct in homogeneous aqueous solution and a 1:2 Michael-Schiff base adduct under two-phase aqueous-organic conditions could be verified by isolation of the respective borohydride-reduced forms. The 1:2 adduct, shown to exist as the cyclic hemiaminal, could represent a stable lysine-based cross-link in certain protein microenvironments.