IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, 52425, Jülich, Germany.
Appl Microbiol Biotechnol. 2019 Aug;103(16):6571-6580. doi: 10.1007/s00253-019-09982-x. Epub 2019 Jun 25.
Pyruvate carboxylase of Corynebacterium glutamicum serves as anaplerotic enzyme when cells are growing on carbohydrates and plays an important role in the industrial production of metabolites derived from the tricarboxylic acid cycle, such as L-glutamate or L-lysine. Previous studies suggested that the enzyme from C. glutamicum is very labile, as activity could only be measured in permeabilized cells, but not in cell-free extracts. In this study, we established conditions allowing activity measurements in cell-free extracts of C. glutamicum and purification of the enzyme by avidin affinity chromatography and gel filtration. Using a coupled enzymatic assay with malate dehydrogenase, V values between 20 and 25 μmol min mg were measured for purified pyruvate carboxylase corresponding to turnover numbers of 160 - 200 s for the tetrameric enzyme. The concentration dependency for pyruvate and ATP followed Michaelis-Menten kinetics with K values of 3.76 ± 0.72 mM and 0.61 ± 0.13 mM, respectively. For bicarbonate, concentrations ≥5 mM were required to obtain activity and half-maximal rates were found at 13.25 ± 4.88 mM. ADP and aspartate inhibited PCx activity with apparent K values of 1.5 mM and 9.3 mM, respectively. Acetyl-CoA had a weak inhibitory effect, but only at low concentrations up to 50 μM. The results presented here enable further detailed biochemical and structural studies of this enzyme.
谷氨酸棒杆菌的丙酮酸羧化酶在细胞以碳水化合物为生长基质时作为固定 CO2 的酶，在三羧酸循环衍生的代谢物（如 L-谷氨酸或 L-赖氨酸）的工业生产中起着重要作用。先前的研究表明，来自谷氨酸棒杆菌的酶非常不稳定，因为其活性只能在通透性细胞中测量，而不能在无细胞提取物中测量。在本研究中，我们建立了在谷氨酸棒杆菌无细胞提取物中测量酶活性并通过亲和层析和凝胶过滤纯化该酶的条件。使用苹果酸脱氢酶偶联酶测定法，对于纯化的丙酮酸羧化酶，V 值在 20 到 25 μmol min mg 之间，对应于四聚体酶的转换数为 160-200 s。对于丙酮酸和 ATP，浓度依赖性遵循米氏动力学，K 值分别为 3.76±0.72 mM 和 0.61±0.13 mM。对于碳酸氢盐，需要≥5 mM 的浓度才能获得活性，半最大速率在 13.25±4.88 mM 时出现。ADP 和天冬氨酸对 PCx 活性具有明显的抑制作用，K 值分别为 1.5 mM 和 9.3 mM。乙酰辅酶 A 具有较弱的抑制作用，但仅在低浓度（高达 50 μM）下有效。这里呈现的结果将使对该酶的进一步详细的生化和结构研究成为可能。
