Centre de Recherche Paul Pascal, Universit de Bordeaux, UPR 8641, Avenue Albert Schweitzer, 33600 Pessac, France.
J Am Chem Soc. 2011 Aug 17;133(32):12801-9. doi: 10.1021/ja204637d. Epub 2011 Jul 25.
Thanks to its insensitivity to dioxygen and to its good catalytic reactivity, and in spite of its poor substrate selectivity, quinoprotein glucose dehydrogenase (PQQ-GDH) plays a prominent role among the redox enzymes that can be used for analytical purposes, such as glucose detection, enzyme-based bioaffinity assays, and the design of biofuel cells. A detailed kinetic analysis of the electrochemical catalytic responses, leading to an unambiguous characterization of each individual steps, seems a priori intractable in view of the interference, on top of the usual ping-pong mechanism, of substrate inhibition and of cooperativity effects between the two identical subunits of the enzyme. Based on simplifications suggested by extended knowledge previously acquired by standard homogeneous kinetics, it is shown that analysis of the catalytic responses obtained by means of electrochemical nondestructive techniques, such as cyclic voltammetry, with ferrocene methanol as a mediator, does allow a full characterization of all individual steps of the catalytic reaction, including substrate inhibition and cooperativity and, thus, allows to decipher the reason that makes the enzyme more efficient when the neighboring subunit is filled with a glucose molecule. As a first practical illustration of this electrochemical approach, comparison of the native enzyme responses with those of a mutant (in which the asparagine amino acid in position 428 has been replaced by a cysteine residue) allowed identification of the elementary steps that makes the mutant type more efficient than the wild type when cooperativity between the two subunits takes place, which is observed at large mediator and substrate concentrations. A route is thus opened to structure-reactivity relationships and therefore to mutagenesis strategies aiming at better performances in terms of catalytic responses and/or substrate selectivity.
由于其对氧气的不敏感性和良好的催化反应活性，尽管其底物选择性较差，但醌蛋白葡萄糖脱氢酶（PQQ-GDH）在可用于分析目的的氧化还原酶中发挥着重要作用，例如葡萄糖检测、基于酶的生物亲和测定和生物燃料电池的设计。鉴于除了通常的乒乓机制之外，底物抑制和酶两个相同亚基之间的协同作用会干扰，对电化学催化响应的详细动力学分析似乎在最初就难以进行，从而无法明确表征每个单独的步骤。基于先前通过标准均相动力学获得的扩展知识所提出的简化，表明通过电化学非破坏性技术（例如循环伏安法）获得的催化响应的分析，使用二茂铁甲醇作为介体，确实可以完全表征催化反应的所有单独步骤，包括底物抑制和协同作用，从而可以解释为什么当相邻亚基充满葡萄糖分子时，酶的效率更高。作为这种电化学方法的第一个实际说明，将天然酶的响应与突变体（其中位置 428 的天冬酰胺氨基酸被半胱氨酸残基取代）的响应进行比较，确定了使突变型比野生型更有效的基本步骤当两个亚基之间发生协同作用时，在大介体和底物浓度下观察到这种情况。因此，开辟了一条通向结构-反应关系的途径，从而开辟了诱变策略，旨在在催化响应和/或底物选择性方面取得更好的性能。
