Spectroscopic detection of chemical intermediates in the reaction of para-substituted benzylamines with bovine serum amine oxidase.
Anaerobic, rapid-scanning stopped-flow spectroscopy has been used to investigate the UV-visible absorbance changes (300-540 nm) that occur in the spectrum of bovine serum amine oxidase during reduction by benzylamine, p-hydroxybenzylamine, and p-methoxybenzylamine. The reaction of enzyme with benzylamine generates detectable relaxations at 310, 340, and 480 nm, which are attributed to the production of reduced cofactor (310 and 480 nm) and to an enzyme-substrate Schiff base complex (340 nm). Additional transients have been observed at 440, 425, and 460 nm with p-hydroxybenzylamine, p-methoxybenzylamine, and p-(N,N-dimethylamino)benzylamine, respectively. These relaxations are ascribed to quinonoid species, formed reversibly from Schiff base complexes between oxidized product and reduced cofactor. With the spectral detection of enzyme-product Schiff base complexes, evidence now exists for each of the postulated chemical intermediates along the reaction path of bovine serum amine oxidase [cf. Hartmann, C., & Klinman, J. P. (1991) Biochemistry 30, 4605]. Anaerobic, single-wavelength stopped-flow data, collected in conjunction with rapid-scanning studies for benzylamine and p-hydroxy-benzylamine, provide approximate rate constants for each of the kinetic processes corresponding to enzyme-substrate Schiff base formation, to enzyme reduction, and to the formation and decay of the quinonoid intermediate.[1]References
- Spectroscopic detection of chemical intermediates in the reaction of para-substituted benzylamines with bovine serum amine oxidase. Hartmann, C., Brzovic, P., Klinman, J.P. Biochemistry (1993) [Pubmed]
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