Ad. Ryabov et al., FERRICENIUM SALTS AS TRUE SUBSTRATES OF GLUCOSE-OXIDASE - A STEADY-STATE KINETIC-STUDY, Applied biochemistry and biotechnology, 61(1-2), 1996, pp. 25-37
The steady-state kinetics of D-glucose oxidation by ferricenium dyes R
Fc(+)PF(6)(-) (R = H, Me, Et, n-Bu, MeCH(2)CMe(2), and Cl) and 1,1' -
Et(2)Fc(+) PF6- catalyzed by glucose oxidase from Aspergillus niger wa
s investigated as a function of RFc(+) and D-glucose concentrations at
pH 6.7, 25 degrees C in the presence of 2% (v/v) Triton X-100. The en
zymatic bleaching is characterized by large steady-state portions on t
he kinetic curves for all ferricenium ions studied. The reaction follo
ws the Michaelis-Menten kinetics demonstrating a high affinity of RFc(
+) toward the active site of reduced glucose oxidase (GO). The reactio
n rate is weakly sensitive to the nature of RFc(+), and the apparent V
-m(app) values decrease only twofold on going from the most to the lea
st reactive salt in the series (1,1'-Et(2)Fc(+) and ClFc(+), respectiv
ely), although their observed redox potentials differ by 160 mV. Remar
kably, the reactivity of RFc(+) does not increase with increasing thei
r oxidative power. The apparent Michaelis constants K-m(app) are also
weakly sensitive to the nature of RFc(+). The profiles for the steady-
state rate vs [HFc(+)] and [D-glucose] were rationalized in terms of t
he ''ping-pong'' mechanism typical of the catalysis by GO. Ferroceneca
rboxylic acid (FcCOOH) appeared to be a competitive inhibitor of GO wi
th the inhibition constant of (3 +/- 1) x 10-(3)M. The pH profile for
the ferricenium fading is bell-shaped with the optimum around 7.5. A s
imple routine for a rapid in situ preparation of the ferricenium dye,
which is ready for spectrophotometric assaying of the GO activity, is
presented. The apparent V-m(app)and K-m(app) values for this substrate
are similar to those for HFc(+) PF6-.