Yomo T, Urabe I, Okada H
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Eur J Biochem. 1991 Sep 15;200(3):759-66. doi: 10.1111/j.1432-1033.1991.tb16242.x.
5-Ethylphenazine-glucose-dehydrogenase-NAD+ conjugate (EP(+)-GlcDH-NAD+) was prepared by linking both poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to glucose dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.8, and that of the NAD+ moieties was 1.2. This conjugate is a semisynthetic enzyme having glucose oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following values of the kinetic constants: for the system with oxygen, the turnover number per subunit is 0.40 s-1 and Km for oxygen is 1.57 mM; and for the system with MTT, the turnover number is 0.11 s-1 and Km for MTT is 0.072 mM. The catalytic cycle of the semisynthetic oxidase has two catalytic steps: reduction of the NAD+ moiety by the active site of the glucose dehydrogenase moiety and oxidation of the NADH moiety by another catalytic site of the ethylphenazine moiety. The apparent intramolecular rate constants of these steps were estimated, and the values are as follows: 0.39 s-1 for the reductions of the NAD+ moiety, 2.2 s-1 and 0.12 s-1 for the oxidation of the NADH moiety in the systems with oxygen and with MTT, respectively, and 3.2 s-1 and 0.18 s-1 for the reduction of the ethylphenazine moiety in the systems with oxygen and with MTT, respectively. On the bases of these results, the following three rate-acceleration mechanisms of the semisynthetic glucose oxidase are discussed: high effective concentration, intramolecular coupling of successive catalytic reactions, and multiple connection between the two kinds of the catalytic sites.
通过将聚乙二醇连接的5-乙基吩嗪和聚乙二醇连接的NAD⁺都连接到葡萄糖脱氢酶上，制备了5-乙基吩嗪-葡萄糖脱氢酶-NAD⁺共轭物（EP(+)-GlcDH-NAD⁺）。结合到每个酶亚基上的乙基吩嗪部分的平均数为0.8，NAD⁺部分的平均数为1.2。这种共轭物是一种半合成酶，以氧气或3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-溴化四氮唑（MTT）作为电子受体时具有葡萄糖氧化酶活性。当氧气或MTT的浓度变化时，氧化酶活性符合米氏方程，动力学常数如下：对于以氧气为体系，每个亚基的周转数为0.40 s⁻¹，氧气的Km为1.57 mM；对于以MTT为体系，周转数为0.11 s⁻¹，MTT的Km为0.072 mM。这种半合成氧化酶的催化循环有两个催化步骤：葡萄糖脱氢酶部分的活性位点将NAD⁺部分还原，以及乙基吩嗪部分的另一个催化位点将NADH部分氧化。估计了这些步骤的表观分子内速率常数，其值如下：在以氧气为体系和以MTT为体系中，NAD⁺部分还原的速率常数分别为0.39 s⁻¹，NADH部分氧化的速率常数分别为氧气体系中的2.2 s⁻¹和MTT体系中的0.12 s⁻¹，以及在以氧气为体系和以MTT为体系中，乙基吩嗪部分还原的速率常数分别为3.2 s⁻¹和0.18 s⁻¹。基于这些结果，讨论了这种半合成葡萄糖氧化酶的以下三种速率加速机制：高效浓度、连续催化反应的分子内偶联以及两种催化位点之间的多重连接。
