FERRICENIUM SALTS AS TRUE SUBSTRATES OF GLUCOSE-OXIDASE - A STEADY-STATE KINETIC-STUDY

Citation
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
Citations number
22
Categorie Soggetti
Biothechnology & Applied Migrobiology",Biology
ISSN journal
02732289
Volume
61
Issue
1-2
Year of publication
1996
Pages
25 - 37
Database
ISI
SICI code
0273-2289(1996)61:1-2<25:FSATSO>2.0.ZU;2-W
Abstract
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-.