Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States of America.
PLoS One. 2012;7(6):e39418. doi: 10.1371/journal.pone.0039418. Epub 2012 Jun 26.
The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, K(m), and the catalytic turnover number, k(cat), of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the K(m) values in the presence and absence of crowding agents have been measured.
细胞质是一个高度浓缩的环境，其中包含多种不同的分子，包括小分子（盐类和代谢物）以及大分子，如核酸、多糖、蛋白质和大型大分子复合物。细胞质环境中的大分子拥挤被认为会影响蛋白质的各种性质，包括底物结合亲和力和酶活性。在这里，我们选择使用合成的拥挤试剂 Ficoll，它常用于模拟细胞质拥挤条件，以研究拥挤对糖酵解酶（即磷酸甘油酸激酶、甘油醛 3-磷酸脱氢酶和酰基磷酸酶）催化特性的影响。我们在不存在和存在拥挤试剂的情况下确定了这些酶的动力学参数。我们发现，这些酶的米氏常数（K m）和催化周转率（k cat）不受拥挤试剂 Ficoll 的存在的影响。我们的结果支持了早期的发现，即某些酶的米氏常数是与细胞内的底物浓度相协调进化的，以允许在双向途径中有效地发挥酶的功能。这一结论进一步得到了对其他 9 种酶的分析的支持，这些酶的 K m 值已经在存在和不存在拥挤试剂的情况下进行了测量。
