Jones G D, Wilson M T
Department of Food Science and Technology, University of Reading, Whiteknights, U.K.
Biochem J. 1988 Dec 15;256(3):713-8. doi: 10.1042/bj2560713.
The reactions between cellobiose and cellobiose oxidase were investigated by stopped-flow spectrophotometry. Under anaerobic conditions rapid reduction of the associated flavin is followed by slower reduction of cytochrome b. The kinetic difference spectra are reported. The rate of flavin reduction depends on the cellobiose concentration (with an apparent second-order rate constant of approx. 10(5) M-1.s-1) but reaches a rate limit of approx. 20 s-1. In contrast, the rate of cytochrome b reduction decreases at high cellobiose concentrations. Kinetic titrations of the flavin and cytochrome b moieties yield the stoichiometries of the separate reactions, i.e. the number of moles of cellobiose needed to fully reduce 1 mole of each redox component. The rate constant for cytochrome b reduction, unlike that for flavin reduction, increased with enzyme concentration, prompting the conclusion that any given cytochrome b centre is reduced preferentially by flavin groups in different molecules rather than by its partner flavin within the same monomer. These data are discussed in the context of a scheme that rationalizes them and accounts for the overall stoichiometry in which three two-electron donors (cellobiose molecules) reduce two three-electron acceptors (the flavin-cytochrome b of cellobiose oxidase).
通过停流分光光度法研究了纤维二糖与纤维二糖氧化酶之间的反应。在厌氧条件下，相关黄素的快速还原之后是细胞色素b的较慢还原。报道了动力学差示光谱。黄素还原速率取决于纤维二糖浓度（表观二级速率常数约为10(5) M-1·s-1），但达到约20 s-1的速率极限。相比之下，在高纤维二糖浓度下细胞色素b的还原速率降低。对黄素和细胞色素b部分进行动力学滴定得出了各单独反应的化学计量，即完全还原1摩尔每种氧化还原成分所需的纤维二糖摩尔数。与黄素还原不同，细胞色素b还原的速率常数随酶浓度增加，这促使得出这样的结论：任何给定的细胞色素b中心优先被不同分子中的黄素基团还原，而不是被同一单体中的伙伴黄素还原。在一个方案的背景下讨论了这些数据，该方案使这些数据合理化并解释了整体化学计量，即三个双电子供体（纤维二糖分子）还原两个三电子受体（纤维二糖氧化酶的黄素 - 细胞色素b）。
