Nakamura A, Urabe I, Okada H
J Biol Chem. 1986 Dec 25;261(36):16792-4.
Polyethylene glycol-bound derivatives of NAD(P) (PEG-NAD(P)) are water-soluble macromolecular coenzymes used in continuous enzyme reactors. These NAD(P) derivatives have good coenzyme activity for many dehydrogenases, but some enzymes such as glucose dehydrogenase (EC 1.1.1.47) show very low activity with these derivatives (less than 0.1% of that for native NAD(P)). In this work, we prepared a covalently linked glucose-dehydrogenase-polyethylene glycol-NAD conjugate (GlcDH-PEG-NAD) and found that the conjugate shows a much higher reaction rate than that of the native enzyme plus PEG-NAD: the ratio of the reaction rates of GlcDH-PEG-NAD and the native enzyme plus PEG-NAD is calculated to be 10,000-fold at the concentrations of the enzyme subunit and NAD moiety of 0.31 and 0.65 microM, respectively; the rate of the conjugate is even higher than that of the native enzyme plus native NAD. This rate acceleration is due to the increase in the effective concentration of NAD moiety ("anchimeric assistance") and demonstrates the potential of covalent linking for improving the interaction between an enzyme and a coenzyme derivative.
聚乙二醇结合的NAD(P)衍生物（PEG-NAD(P)）是用于连续酶反应器的水溶性大分子辅酶。这些NAD(P)衍生物对许多脱氢酶具有良好的辅酶活性，但一些酶，如葡萄糖脱氢酶（EC 1.1.1.47），对这些衍生物的活性非常低（低于天然NAD(P)活性的0.1%）。在本研究中，我们制备了一种共价连接的葡萄糖脱氢酶-聚乙二醇-NAD缀合物（GlcDH-PEG-NAD），发现该缀合物的反应速率比天然酶加PEG-NAD的反应速率高得多：在酶亚基和NAD部分浓度分别为0.31和0.65微摩尔时，GlcDH-PEG-NAD与天然酶加PEG-NAD的反应速率之比经计算为10000倍；缀合物的速率甚至高于天然酶加天然NAD的速率。这种速率加速是由于NAD部分有效浓度的增加（“邻基协助”），并证明了共价连接在改善酶与辅酶衍生物之间相互作用方面的潜力。
