期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 100, 期 1, 页码 62-67出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.252644599
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资金
- NIGMS NIH HHS [GM 20709, R01 GM025765, GM 25765, R37 GM025765, F32 GM020709] Funding Source: Medline
Two prototropic forms of glucose oxidase undergo aerobic oxidation reactions that convert FADH(-) to FAD and form H2O2 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 (283323 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.
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