Kinetic studies, mechanism, and substrate specificity of amadoriase I from Aspergillus sp.
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 O(2) as substrates in 10 mM Tris HCl, pH 7.9, 4 degrees C. Using both substrate analogues and fast kinetic techniques, the active configuration of the substrate was found to be the beta-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 turnover rate, 14.4 s(-1), suggesting that it is not involved in the catalytic cycle and that reoxidation of the reduced enzyme occurs in the E(red)-product complex. In the oxidative half-reaction, the reduced flavin is oxidized by O(2) in a single phase. The observed rate constant has a linear dependence on oxygen concentration, giving a bimolecular rate constant of 4.9 x 10(4) M(-1) s(-1) in the absence 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, giving values of +48 and -52 mV for the oxidized enzyme/anionic semiquinone and anionic semiquinone/reduced enzyme couples, respectively.
Wu
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X
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Palfey
B A
BA
Mossine
V V
VV
Monnier
V M
VM
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D001230:Aspergillus / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D019270:Fructosamine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D019270:Fructosamine; D006861:Hydrogen Peroxide; D000594:Amino Acid Oxidoreductases; C103012:amadoriase; D010100:Oxygen