Stopped-flow spectrophotometric characterization of enzymic reaction intermediates in the anaerobic reduction of pig-plasma benzylamine oxidase by amine substrates.
Reduction of benzylamine oxidase by p-methoxybenzylamine under anaerobic conditions leads to biphasic absorbance changes at 470 nm. These reflect the intermediate formation of an enzyme substrate complex with spectral properties different from those of native enzyme and fully reduced enzyme. The spectrally modified enzyme-substrate complex exhibits a broad difference absorption band centered around 360 nm. The transient accumulation of this intermediate during reaction can be conveniently followed by stopped-flow techniques at wavelengths between 320 and 360 nm, where contributions from the subsequent reduction of the enzymic 470-nm chromophore are of minor significance. 2. Analogous intermediates exhibiting similar absorption spectra seem to be formed on reduction of the enzyme by benzylamine and other amine substrates which were tested. Substitution of benzylamine as the reducing substrate by [alpha, alpha-2H]benzylamine results in a decreased accumulation of the spectrally modified intermediate. This indicates that its formation is preceded by deprotonation of the alpha-carbon of the amine substrate. 3. Circular dichroism spectra of benzylamine oxidase exhibit a positive band at 360 nm, lending support to the previous conclusion that benzylamine oxidase is a pyridoxal enzyme. Formation of the spectrally modified enzyme-substrate complex then most likely reflects the prototropic shift converting an amine-pyridoxal Schiff-base obtained by rapid pre-equilibration between enzyme and substrate into an aldehyde-pyridoxamine Schiff-base.
D000588:Amines / Q000378:metabolism*; D000818:Animals; D001595:Benzylamine Oxidase / Q000097:blood / Q000378:metabolism*; D001596:Benzylamines / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008995:Monoamine Oxidase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013552:Swine
D000588:Amines; D001596:Benzylamines; D003903:Deuterium; D001595:Benzylamine Oxidase; D008995:Monoamine Oxidase