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

Amadoriase is a flavoenzyme that catalyzes the oxidative deglycation of Ama dori products (fructosyl amino acids or aliphatic amines) to yield free ami ne, glucosone, and hydrogen peroxide. The mechanism of action of amadoriase I from Aspergillus sp. has been investigated by stopped-flow kinetic studi es using fructosyl propylamine and O-2 as substrates in 10 mM Tris HCl, pH 7.9, 4 degreesC. Using both substrate analogues and fast kinetic techniques , the active configuration of the substrate was found to be the beta -pyran ose form. Stopped-flow studies showed that the reductive half-reaction is t riphasic and generates intermediates that absorb at long wavelengths and is consistent either with (i) the reaction of the substrate with the flavin f ollowed by iminium, deprotonation or hydrolysis and then product release or with (ii) the formation of flavin reduction intermediates (carbanion equiv alents or adducts), followed by product release. The rate of product releas e after flavin reduction is lower than the aerobic turnover rate, 14.4 s(-1 ), suggesting that it is not involved in the catalytic cycle and that reoxi dation of the reduced enzyme occurs in the E-red-product complex. In the ox idative half-reaction, the reduced flavin is oxidized by O-2 in a single ph ase. The observed rate constant has a linear dependence on oxygen concentra tion, giving a bimolecular rate constant of 4.9 x 10(4) M-1 s(-1) in the ab sence of product, and 3.6 x 10(4) M-1 s(-1) when the product is bound. The redox potentials of amadoriase have been measured at pH 7.0, 25 degrees, gi ving values of +48 and -52 mV for the oxidized enzyme/anionic semiquinone a nd anionic semiquinone/reduced enzyme couples, respectively.