The Glucose Oxidase Mechanism

摘要: Abstract The pH dependence of the steady state parameters glucose oxidase (EC 1.1.3.4, from Aspergillus niger) reaction was determined by O2-monitored experiments over entire range 3 to 10 at 25°, with d-glucose as substrate. data were fitted a three-parameter rate equation and significance examined stopped flow half-reaction turnover measurements extremes used. major conclusions these studies can be summarized follows. 1. At low pH, in presence halide, maximum turn-over number (kcat) is entirely flavin reduction (k2) reductive half-reaction. Furthermore, substrate combines only an unprotonated form oxidized enzyme represented H+ E0 (K1)/⇄/(H+) + S (k1)/⇄/(k-1) - (k2)/→ Er δ-lactone Since kcat k2 are both specifically decreased halides values, it probable that also limited absence halide. absorption spectrum indistinguishable E0. This finding, together fact removal 1-hydrogen ratelimiting process consistent hydride transfer mechanism flavin-glucose adduct which this relatively unstable never accumulates significantly kinetic intermediate. 2. importance limiting first order diminishes raised. Thus, breakdown species enzyme, E'0, oxidative 10.0 214 sec-1, whereas has value 800 sec-1. 3. reduced exists two kinetically significant states ionization, Er-. rapid reoxidation O2, regenerate E0, predominant values less than 7. greater 7, much Er- leading formation E'0-, becomes increasingly important. E'0- unreactive conversion protonated principally governs We present complete scheme describing effects discuss possible chemical E'0.