Abstract
The observation that aliphatic diamines become poor substrates as the carbon chain length decreases and that ethylenediamine, the shortest diamine, is an irreversible inhibitor of lysyl oxidase led to the investigation of the mechanism of inhibition by ethylenediamine. The cis but not the trans isomer of 1,2-diaminocyclohexane was also a potent irreversible inhibitor of lysyl oxidase, consistent with the interaction of both amino groups of vicinal diamines with an enzyme moiety. Both cis-1,2-diaminocyclohexane and ethylenediamine but not trans-1,2-diaminocyclohexane markedly perturbed the spectrum of free pyrroloquinoline quinone (PQQ), a covalently linked form of which is the carbonyl cofactor of lysyl oxidase. cis-1,2-Diaminocyclohexane also induced similar changes in the spectrum of lysyl oxidase. The perturbations of the spectra of PQQ or of lysyl oxidase by cis-1,2-diaminocyclohexane or ethylenediamine as well as the development of irreversible inhibition of the enzyme by cis-1,2-diaminocyclohexane or ethylenediamine were all markedly reduced under anaerobic conditions. Moreover, approximately 1 mol of H2O2 was released per mol of PQQ or lysyl oxidase upon aerobic incubation with cis-1,2-diaminocyclohexane, while approximately 2 mol of 3H+ were released from cis-[1,2-3H] 1,2-diaminocyclohexane per mol of PQQ or lysyl oxidase under corresponding conditions. A proposal for the mechanism of inhibition of lysyl oxidase by vicinal diamines is presented which involves limited oxidation of the diamine linked to PQQ at the active site so that the PQQ-diamine complex is finally stabilized by a conjugated 6-membered ring.
Highlights
The observationthat aliphatic diaminesbecome poor for lysyl oxidase have chemotherapeuticpotential as antisubstrates as the carbon chain length decreases and fibrotic agents
It hasbeen reported previously that various bifunctional compounds, including aliphatic diamines, disulfhydryl compounds, and compounds containing both sulfhydryl and Diaminocyclohexane induced similar changes in amine substituents inhibit lysyl oxidase, and it was suggested the spectrumof lysyl oxidase.The perturbationsof the that theirreversible inhibition noted withsome of these may spectra of PQQ or of lysyl oxidase by cis-1,Z-diami- reflect adduct formation witthhe carbonyl cofactor which had nocyclohexane or ethylenediamine as well as the de- not been identified at thattime [4].In thepresent study, we velopment of irreversible inhibitionof the enzyme by have investigated the reaction of lysyl oxidase with linear and cis- 1,Z-diaminocyclohexane or ethylenediamine were cyclic aliphatic diamines notingevidence for interaction with allmarkedly reduced underanaerobic conditions. the PQQ' cofactor and observing that a sterically restricted
Steady-statekinetic analyses of inhibitor mechanisms were restricted to concentrations not greater than 1.2 pM cis-1,2-diaminocyclohexaneA. nalogous considerations were taken into account in the analyses of the inhibition by ethylenediamine
Summary
1,Z-diaminocyclohexane per mol of PQQ o r lysyl oxidase under corresponding conditions. Among the copper-dependent amineoxidases, lysyl oxidase similar and the substrate specificities and inhibition profiles of the is unique by virtue of its ability to catalyze the oxidative variants appear to be virtually the same indicating that thecatalytic deamination of peptidyl lysine inelastin and collagen to mechanism is likely to be the same for each of these enzyme forms peptidyl a-aminoadipic-hemialdehyde. This peptidyl aldehyde can condense with other aldehyde and unreacted lysine residues to generate the covalent cross-linkages which underlie the insolubility of these connective tissue proteins.In view of the central role of this enzyme in connective tissue fiber [6]. The enzyme was assayed against alkyl mono- and diamines by a peroxidase-coupledfluorescence method at 55 "C [9].The reaction mixtures contained 40pgof horseradish peroxidase, 0.7 mM
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