title: Catalysis of electron transfer during activation of O2 by the flavoprotein glucose oxidase.
abstract: 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.
authors.affiliation: Department of Chemistry and, University of California, Berkeley, 94720, USA.
authors.forename: Justine P
authors.initials: JP
authors.lastname: Roth
authors.forename: Judith P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: 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
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005949:Glucose Oxidase