Catalysis of electron transfer during activation of O2 by the flavoprotein glucose oxidase |
| |
Authors: | Roth Justine P Klinman Judith P |
| |
Affiliation: | Department of Chemistry and, University of California, Berkeley, 94720, USA. |
| |
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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|