Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity
Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity
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DOI：
10.1093/jb/mvh089
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发表时间：
2004-06-01
影响因子：
2.7
通讯作者：
Horiike, K
中科院分区：
文献类型：
--
作者：
Ishida, T;Tanaka, H;Horiike, K
来自恶臭假单胞菌mt-2（Mpc）的邻苯二酚2，3-双加氧酶[EC 1.13.11.2]催化邻苯二酚的外二醇裂解，生成2-羟基粘康酸半醛。在25 ℃和pH6.5或7.5条件下，测定了8种C3/C4取代邻苯二酚的邻苯二酚底物Km值（K-mA）和O-2 Km值（K-mO 2）以及催化常数（k（cat））。测定了11种儿茶酚的第一个pK（a）值（pK（1））（pK（1）= 7.26-9.47），与Hammett取代基常数相关，吸电子取代基显著稳定了游离儿茶酚的单阴离子物种。Mpc优选在C3或C4位置具有非离子取代基的儿茶酚。3-苯基邻苯二酚（一种联苯）被裂解，而4-叔丁基邻苯二酚则不裂解。k（cat）/K-mA（底物特异性常数）的对数与pK（1）呈良好的线性关系，但4-卤代儿茶酚除外。k（cat）/K-mO_2的对数与pK（1）呈良好的线性关系，但3-苯基邻苯二酚除外。这些结果表明，邻苯二酚与Mpc活性中心的结合，随后的O-2结合，以及结合O-2的活化都对取代基的电子效应敏感。然而，k（cat）与pK（1）无显著相关性。本研究区分清楚的电子和儿茶酚底物的立体效应的反应性MPC，并提供了重要的洞察力的机械基础为广大范围的底物特异性的extradiol双加氧酶。
Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The Km values for the catecholic substrate (K-mA) and O-2 (K-mO2), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25degreesC and pH 6.5 or 7.5. The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tent-butylcatechol was not. The logarithm of k(cat)/K-mA (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols. The logarithm of k(cat)/K-mO2 showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O-2 binding, and the activation of the bound O-2 are all sensitive to electronic effects of the substituents. However, k(cat) did not correlate significantly with pK(1). The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.