Tao Zhimin, Raffel Ryan A, Souid Abdul-Kader, Goodisman Jerry
Department of Chemistry, Syracuse University, Syracuse, New York 13244, USA.
Biophys J. 2009 Apr 8;96(7):2977-88. doi: 10.1016/j.bpj.2008.11.071.
The kinetics of the glucose oxidase-catalyzed reaction of glucose with O2, which produces gluconic acid and hydrogen peroxide, and the catalase-assisted breakdown of hydrogen peroxide to generate oxygen, have been measured via the rate of O2 depletion or production. The O2 concentrations in air-saturated phosphate-buffered salt solutions were monitored by measuring the decay of phosphorescence from a Pd phosphor in solution; the decay rate was obtained by fitting the tail of the phosphorescence intensity profile to an exponential. For glucose oxidation in the presence of glucose oxidase, the rate constant determined for the rate-limiting step was k = (3.0 +/- 0.7) x 10(4) M(-1) s(-1) at 37 degrees C. For catalase-catalyzed H2O2 breakdown, the reaction order in [H2O2] was somewhat greater than unity at 37 degrees C and well above unity at 25 degrees C, suggesting different temperature dependences of the rate constants for various steps in the reaction. The two reactions were combined in a single experiment: addition of glucose oxidase to glucose-rich cell-free media caused a rapid drop in [O2], and subsequent addition of catalase caused [O2] to rise and then decrease to zero. The best fit of [O2] to a kinetic model is obtained with the rate constants for glucose oxidation and peroxide decomposition equal to 0.116 s(-1) and 0.090 s(-1) respectively. Cellular respiration in the presence of glucose was found to be three times as rapid as that in glucose-deprived cells. Added NaCN inhibited O2 consumption completely, confirming that oxidation occurred in the cellular mitochondrial respiratory chain.
葡萄糖氧化酶催化葡萄糖与氧气反应生成葡萄糖酸和过氧化氢的动力学过程，以及过氧化氢酶辅助过氧化氢分解产生氧气的过程，已通过氧气消耗或产生的速率进行了测量。通过测量溶液中钯磷光体磷光的衰减来监测空气饱和磷酸盐缓冲盐溶液中的氧气浓度；衰减速率通过将磷光强度曲线的尾部拟合为指数函数来获得。对于在葡萄糖氧化酶存在下的葡萄糖氧化，在37℃时确定的限速步骤的速率常数为k = (3.0±0.7)×10⁴ M⁻¹ s⁻¹。对于过氧化氢酶催化的过氧化氢分解，在37℃时[H₂O₂]的反应级数略大于1，在25℃时远大于1，这表明反应中各个步骤的速率常数对温度的依赖性不同。在单个实验中将这两个反应结合起来：向富含葡萄糖的无细胞培养基中添加葡萄糖氧化酶会导致[O₂]迅速下降，随后添加过氧化氢酶会使[O₂]先上升然后降至零。将[O₂]与动力学模型进行最佳拟合时，葡萄糖氧化和过氧化物分解的速率常数分别为0.116 s⁻¹和0.090 s⁻¹。发现在有葡萄糖存在的情况下细胞呼吸比在缺乏葡萄糖的细胞中快三倍。添加的NaCN完全抑制了氧气消耗，证实氧化发生在细胞线粒体内呼吸链中。
