Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis
Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis
复制标题
DOI：
10.1016/j.abb.2016.06.009
复制
发表时间：
2016-08-15
影响因子：
3.9
通讯作者：
Pierce, Brad S.
中科院分区：
文献类型：
--
作者：
Crowell, Joshua K.;Sardar, Sinjinee;Pierce, Brad S.
3-棕色固氮菌（Azotobacter vinelandii）的巯基丙酸双加氧酶（Av MDO）是一种非血红素单核铁酶，其催化3-巯基丙酸（3 mpa）的O-2依赖性氧化以产生3-亚磺基丙酸（3spa）。除了一个例外，MDO的活性位点残基与细菌半胱氨酸双加氧酶（CDO）相同。具体地，MDO中的CDO Arg-残基（R50）被Gln（Q67）替代。尽管有这种微小的活性位点干扰，但与CDO相比，Av MDO的底物特异性更为宽松。为了研究Av MDO催化中化学和非化学事件的相对时序，使用两种不同的底物[3 mpa和L-半胱氨酸（cys）]测量稳态动力学参数（k（cat）和k（cat）/K-M）和粘度效应的pH/D依赖性。这些反应中Av MDO的pL依赖性活性可以合理化，假设双质子酶模型，其中存在三种离子形式的酶[阳离子，E（z+1）;中性，E-z;和阴离子，E（z-1）]。观察到的每个基板的活动似乎是占主导地位的酶活性位点内的静电相互作用。鉴于MDO和更广泛的特点哺乳动物CDO之间的相似性，保守的“催化三联体”的作用提出了一个初步的模型。(C)2016 Elsevier Inc. All rights reserved.
3-mercaptopropionate dioxygenase from Azotobacter vinelandii (Av MDO) is a non-heme mononuclear iron enzyme that catalyzes the O-2-dependent oxidation of 3-mercaptopropionate (3mpa) to produce 3-sulfinopropionic acid (3spa). With one exception, the active site residues of MDO are identical to bacterial cysteine dioxygenase (CDO). Specifically, the CDO Arg-residue (R50) is replaced by Gln (Q67) in MDO. Despite this minor active site perturbation, substrate-specificity of Av MDO is more relaxed as compared to CDO. In order to investigate the relative timing of chemical and non-chemical events in Av MDO catalysis, the pH/D-dependence of steady-state kinetic parameters (k(cat) and k(cat)/K-M) and viscosity effects are measured using two different substrates [3mpa and L-cysteine (cys)]. The pL-dependent activity of Av MDO in these reactions can be rationalized assuming a diprotic enzyme model in which three ionic forms of the enzyme are present [cationic, E(z+1); neutral, E-z; and anionic, E(z-1)]. The activities observed for each substrate appear to be dominated by electrostatic interactions within the enzymatic active site. Given the similarity between MDO and the more extensively characterized mammalian CDO, a tentative model for the role of the conserved 'catalytic triad' is proposed. (C) 2016 Elsevier Inc. All rights reserved.