Bull C, Ballou D P, Otsuka S
J Biol Chem. 1981 Dec 25;256(24):12681-6.
The reactions of protocatechuate dioxygenase (protocatechuate:oxygen 3,4-oxidoreductase, EC 1.13.11.3) with substrates and oxygen have been studied at 4 degrees C using rapid kinetic techniques. In this study, two oxygenated intermediates were kinetically and spectrally characterized. The rate of oxygen addition to the enzyme-substrate complex was determined to be 5 X 10(5) M-1 s-1. This oxygenated complex rapidly converts (450 s-1) to another spectrally identifiable compound which then breaks down into free enzyme and the product, beta-carboxy-cis,cis-muconic acid. To carry out these experiments properly, it was necessary to use hyperbaric oxygen, thus obtaining approximately 6 mM final concentration of oxygen. The procedure is described in this work. It was also shown that the normally slow substrate, dihydroxyphenyl propionate, when reacted with the enzyme and oxygen, rapidly forms two sequential intermediates. However, the second intermediate was not spectrally the same as that observed with protocatechuate, but was more like that of enzyme-product complexes. Similar, although less extensive, studies using the protocatechuate dioxygenase from Pseudomonas aeruginosa showed that very analogous behavior occurred with both substrates as did with the Pseudomonas putida enzyme.
已在4℃下使用快速动力学技术研究了原儿茶酸双加氧酶（原儿茶酸：氧3,4-氧化还原酶，EC 1.13.11.3）与底物及氧气的反应。在本研究中，对两种氧化中间体进行了动力学和光谱表征。确定酶-底物复合物加氧的速率为5×10⁵ M⁻¹ s⁻¹。这种氧化复合物迅速转化（450 s⁻¹）为另一种可通过光谱识别的化合物，然后分解为游离酶和产物β-羧基-顺,顺-粘康酸。为了正确进行这些实验，有必要使用高压氧，从而获得约6 mM的最终氧气浓度。本文描述了该方法。还表明，通常反应较慢的底物二羟基苯丙酸与酶和氧气反应时，会迅速形成两个连续的中间体。然而，第二个中间体在光谱上与原儿茶酸观察到的不同，而更类似于酶-产物复合物。使用铜绿假单胞菌的原儿茶酸双加氧酶进行的类似但规模较小的研究表明，两种底物的行为与恶臭假单胞菌的酶非常相似。
