Papezová Katerina, Nemec Tomás, Chaloupková Radka, Glatz Zdenek
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
J Chromatogr A. 2007 May 25;1150(1-2):327-31. doi: 10.1016/j.chroma.2006.09.022. Epub 2006 Sep 29.
Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine-hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate--1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (K(M)) as well as the substrate inhibition constant (K(SI)). The value of K(M) and K(SI) obtained were 7.7+/-2.5 mM and 1.1+/-0.4 mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.
底物抑制是酶动力学中的一种常见现象。我们首次在此报告通过电泳介导微分析（EMMA）方法与部分填充技术相结合对其进行的研究。在此设置中，毛细管的一部分填充最适合酶促反应的缓冲液，而毛细管的其余部分填充最适合底物和产物分离的背景电解质。对于本研究中选择的模型酶卤代烷脱卤酶，酶促反应在20 mM甘氨酸缓冲液（pH 8.6）中进行，而20 mMβ-丙氨酸 - 盐酸缓冲液（pH 3.5）用作背景电解质，并在200 nm处进行直接检测。整个研究是针对难溶性溴化底物——1,2 - 二溴乙烷进行的。因此，首先有必要在酶和底物的浓度之间找到折衷方案，既要保持测定的足够灵敏度，又要同时保证可达到的底物溶解度。通过所开发的EMMA方法，我们能够确定米氏常数（K(M)）以及底物抑制常数（K(SI)）。获得的K(M)和K(SI)值分别为7.7±2.5 mM和1.1±0.4 mM。1,2 - 二溴乙烷对卤代烷脱卤酶的底物抑制作用观察结果与先前的文献数据一致。
