Sato A, Takagi K, Kano K, Kato N, Duine J A, Ikeda T
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Biochem J. 2001 Aug 1;357(Pt 3):893-8. doi: 10.1042/0264-6021:3570893.
Spectroelectrochemical studies were performed on the interaction between Ca(2+) and pyrroloquinoline quinone (PQQ) in soluble glucose dehydrogenase (sGDH) and in the free state by applying a mediated continuous-flow column electrolytic spectroelectrochemical technique. The enzyme forms used were holo-sGDH (the holo-form of sGDH from Acinetobacter calcoaceticus) and an incompletely reconstituted form of this, holo-X, in which the PQQ-activating Ca(2+) is lacking. The spectroelectrochemical and ESR data clearly demonstrated the generation of the semiquinone radical of PQQ in holo-sGDH and in the free state in the presence of Ca(2+). In contrast, in the absence of Ca(2+) no semiquinone was observed, either for PQQ in the free state (at pH 7.0) or in the enzyme (holo-X). Incorporation of Ca(2+) into the active site of holo-X, yielding holo-sGDH, caused not only stabilization of the semiquinone form of PQQ but also a negative shift (of 26.5 mV) of the two-electron redox potential, indicating that the effect of Ca(2+) is stronger on the oxidized than on the reduced PQQ. Combining these data with the observations on the kinetic and chemical mechanisms, it was concluded that the strong stimulating effect of Ca(2+) on the activity of sGDH can be attributed to facilitation of certain kinetic steps, and not to improvement of the thermodynamics of substrate oxidation. The consequences of this conclusion are discussed for the oxidative as well as for the reductive part of the reaction of sGDH.
采用介导连续流动柱电解光谱电化学技术，对可溶性葡萄糖脱氢酶（sGDH）中以及游离状态下的Ca(2+)与吡咯喹啉醌（PQQ）之间的相互作用进行了光谱电化学研究。所使用的酶形式为全酶-sGDH（来自乙酸钙不动杆菌的sGDH的全酶形式）及其不完全重构形式全酶-X，其中缺乏PQQ激活所需的Ca(2+)。光谱电化学和电子顺磁共振数据清楚地表明，在Ca(2+)存在下，全酶-sGDH和游离状态下的PQQ会生成半醌自由基。相比之下，在没有Ca(2+)的情况下，无论是游离状态（pH 7.0）的PQQ还是酶（全酶-X）中，均未观察到半醌。将Ca(2+)掺入全酶-X的活性位点，生成全酶-sGDH，不仅使PQQ的半醌形式得以稳定，而且使双电子氧化还原电位负移（26.5 mV），这表明Ca(2+)对氧化态PQQ的作用比对还原态PQQ的作用更强。将这些数据与动力学和化学机制的观察结果相结合，得出结论：Ca(2+)对sGDH活性的强烈刺激作用可归因于某些动力学步骤的促进，而不是底物氧化热力学的改善。针对sGDH反应的氧化部分和还原部分，讨论了这一结论的影响。
