Yomo T, Sawai H, Urabe I, Okada H
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Eur J Biochem. 1989 Feb 1;179(2):299-305. doi: 10.1111/j.1432-1033.1989.tb14555.x.
5-Ethylphenazine-poly(ethylene glycol)-NAD+ conjugate (EP+-PEG-NAD+) was prepared by linking 1-(3-carboxypropyloxy)-5-ethylphenazine (I) to poly(ethylene glycol)-bound NAD+ (PEG-NAD+) and its kinetic properties were studied. As a reference compound, poly(ethylene glycol)-bound 5-ethylphenazine derivative (III) was also prepared and the effects of poly(ethylene glycol) on the reaction rate of the 5-ethylphenazine moiety with NADH was investigated. The second-order rate constant, k1, of the reaction of III with NADH is 2.78 mM-1 s-1 and is about 1.7 times that of 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine (II) with NADH. A similar effect of the attached poly(ethylene glycol) was observed for the reaction of PEG-NADH with I or II. The second-order rate constants, k2 and k3, of the reactions of the reduced form of III with oxygen and with 3-(4',5'-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium ion, respectively, were k2 = 1.22 mM-1 s-1 and k3 = 32 mM-1 s-1; the k2 value is not changed but the k3 value is decreased by the attachment of the polymer. EP+-PEG-NAD+ works as a unique catalyst having an intramolecular reaction step within its turnover cycle in a coupled multi-step reaction system containing malate dehydrogenase, malate, EP+-PEG-NAD+, a tetrazolium salt and oxygen. The first-order rate constant, k4, of the intramolecular reaction was 1.1 s-1. The effects of the covalent linking of the 5-ethylphenazine and the NAD+ moieties were estimated by comparing the value of k4 with that of k1 for the reaction of III with NADH; the effective concentration of the NADH moiety for the 5-ethylphenazine moiety on the same EP+-PEG-NADH molecule (or vice versa) was calculated to be 0.40 mM from the ratio of k4/k1. The values of the rate constants in the coupled multi-step reaction system enable us to understand the dynamic features of the system and the characteristics of EP+-PEG-NAD+ as a catalyst are discussed.
通过将1-(3-羧基丙氧基)-5-乙基吩嗪（I）与聚乙二醇结合的NAD⁺（PEG-NAD⁺）相连制备了5-乙基吩嗪-聚乙二醇-NAD⁺共轭物（EP⁺-PEG-NAD⁺），并研究了其动力学性质。作为参考化合物，还制备了聚乙二醇结合的5-乙基吩嗪衍生物（III），并研究了聚乙二醇对5-乙基吩嗪部分与NADH反应速率的影响。III与NADH反应的二级速率常数k₁为2.78 mM⁻¹ s⁻¹，约为1-(3-乙氧羰基丙氧基)-5-乙基吩嗪（II）与NADH反应速率常数的1.7倍。对于PEG-NADH与I或II的反应，观察到连接的聚乙二醇有类似的影响。III的还原形式与氧气和3-(4',5'-二甲基噻唑-2-基)-2,5-二苯基四氮唑离子反应的二级速率常数k₂和k₃分别为k₂ = 1.22 mM⁻¹ s⁻¹和k₃ = 32 mM⁻¹ s⁻¹；聚合物的连接使k₂值不变，但k₃值降低。在包含苹果酸脱氢酶、苹果酸、EP⁺-PEG-NAD⁺、四氮唑盐和氧气的耦合多步反应体系中，EP⁺-PEG-NAD⁺作为一种独特的催化剂，在其周转循环中有一个分子内反应步骤。分子内反应的一级速率常数k₄为1.1 s⁻¹。通过比较k₄与III与NADH反应的k₁值，估计了5-乙基吩嗪和NAD⁺部分共价连接的影响；根据k₄/k₁的比值，计算出同一EP⁺-PEG-NADH分子上5-乙基吩嗪部分的NADH部分的有效浓度为0.40 mM。耦合多步反应体系中的速率常数数值使我们能够理解该体系的动态特征，并讨论了EP⁺-PEG-NAD⁺作为催化剂的特性。
