Catalysis of electron transfer during activation of O2 by the flavoprotein glucose oxidase.
Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH(-) to FAD and form H(2)O(2) as a product. Limiting rate constants of k(cat)K(M)(O(2)) = (5.7 +/- 1.8) x 10(2) M(-1).s(-1) and k(cat)K(M)(O(2)) = (1.5 +/- 0.3) x 10(6) M(-1).s(-1) are observed at high and low pH, respectively. Reactions exhibit oxygen-18 kinetic isotope effects but no solvent kinetic isotope effects, consistent with mechanisms of rate-limiting electron transfer from flavin to O(2). Site-directed mutagenesis studies reveal that the pH dependence of the rates is caused by protonation of a highly conserved histidine in the active site. Temperature studies (283-323 K) indicate that protonation of His-516 results in a reduction of the activation energy barrier by 6.0 kcal.mol(-1) (0.26 eV). Within the context of Marcus theory, catalysis of electron transfer is attributed to a 19-kcal.mol(-1) (0.82 eV) decrease in the reorganization energy and a much smaller 2.2-kcal.mol(-1) (0.095 eV) enhancement of the reaction driving force. An explanation is advanced that is based on changes in outer-sphere reorganization as a function of pH. The active site is optimized at low pH, but not at high pH or in the H516A mutant where rates resemble the uncatalyzed reaction in solution.
Roth
Justine P
JP
Department of Chemistry and, University of California, Berkeley, 94720, USA.
Klinman
Judith P
JP
D001234:Aspergillus niger / Q000201:enzymology; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010101:Oxygen Consumption; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D017931:DNA Primers; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005949:Glucose Oxidase