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Mechanistic studies of formate oxidase from Aspergillus oryzae: A novel member of the glucose-Methanol-choline oxidoreductase enzyme superfamily that oxidizes carbon acids
- Robbins, John M., Bommarius, Andreas S., Gadda, Giovanni
- Archives of biochemistry and biophysics 2018 v.643 pp. 24-31
- Aspergillus oryzae, alcohols, catalytic activity, cleavage (chemistry), deuterium, formates, isomerization, isotopes, oxidoreductases, pH, solvents, ultracentrifugation, viscosity
- Formate oxidase (FOX) from Aspergillus oryzae is the only GMC member that oxidizes a carbon acid rather than alcohols; thus, its catalytic mechanism may be different from that of other GMC members. We have used pH, solvent viscosity, and deuterium kinetic isotope effects, to investigate the catalytic mechanism of FOX. The enzyme followed a Bi-Bi sequential steady-state kinetic mechanism. The kcat value was pH-independent between pH 2.8 and 6.8, suggesting a lack of ionizable groups in kinetic step(s) that limit the overall turnover of the enzyme. The kcat/Kformate value decreased from a value of 10,000 M⁻¹s⁻¹ at low pH with a pKa value of 4.4, consistent with the requirement of a protonated group for substrate binding. An inverse viscosity dependence on the kcat/Kformate value indicated an isomerization of the Michaelis complex. The kcat/Koxygen value was 340,000 M⁻¹s⁻¹ and pH independent up to pH 6.0. The ᴰkcat and ᴰ(kcat/Kformate) values were 2.5 and 1.9, respectively, indicating that substrate CH bond cleavage is rate-limiting for FOX catalysis. Analytical ultracentrifugation indicated a concentration dependence of the oligomeric state of FOX. The ᵃᵖᵖkred,H value was ∼75% that of kcat,H, indicating that the anaerobic reduction of FOX was dependent on the oligomeric state of FOX.