Kinetic studies, mechanism and substrate specificity of amadoriase I from Aspergillus sp.

Abstract

Amadoriase is a flavoenzyme that catalyzes the oxidative deglycation of Amadori products (fructosyl amino acids or aliphatic amines) to yield free amine, glucosone, and hydrogen peroxide. The mechanism of action of amadoriase I from Aspergillus sp. has been investigated by stopped-flow kinetic studies using fructosyl propylamine and O2 as substrates in 10 mM Tris HCl, pH 7.9, 4 °C. Using both substrate analogues and fast kinetic techniques, the active configuration of the substrate was found to be the β-pyranose form. Stopped-flow studies showed that the reductive half-reaction is triphasic and generates intermediates that absorb at long wavelengths and is consistent either with (i) the reaction of the substrate with the flavin followed by iminium deprotonation or hydrolysis and then product release or with (ii) the formation of flavin reduction intermediates (carbanion equivalents or adducts), followed by product release. The rate of product release after flavin reduction is lower than the aerobic turnov...