Zomer A W, Allert S, Chevalier N, Callens M, Opperdoes F R, Michels P A
Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Catholic University of Louvain, Brussels, Belgium.
Biochim Biophys Acta. 1998 Jul 28;1386(1):179-88. doi: 10.1016/s0167-4838(98)00095-8.
The Trypanosoma brucei phosphoglycerate kinase (PGK) glycosomal and cytosolic isoenzymes have been overexpressed in Escherichia coli and purified to near-homogeneity. Both enzymes were similar to the corresponding natural proteins with respect to their physicochemical and kinetic properties. In addition, a mutant of the glycosomal PGK lacking the 20 amino acid long C-terminal extension was overexpressed and purified. Various properties of this truncated glycosomal PGK were examined and it was found that in some aspects the protein behaved quite differently when compared with its natural counterpart. This was notably the case for the apparent Km for 3-phosphoglyceric acid, its sensitivity to inhibitors and its response to salts and guanidine HCl. However, its Vmax was found to be similar to that of the natural glycosomal PGK. These results suggest that the changes in the C-terminus caused a conformational change effecting the 3-phosphoglyceric acid binding site located at the N-terminal domain of the protein.
布氏锥虫磷酸甘油酸激酶（PGK）的糖体同工酶和胞质同工酶已在大肠杆菌中过表达，并纯化至接近均一。这两种酶在物理化学和动力学性质方面均与相应的天然蛋白质相似。此外，缺少20个氨基酸长的C末端延伸的糖体PGK突变体也被过表达并纯化。对这种截短的糖体PGK的各种性质进行了研究，发现与天然对应物相比，该蛋白质在某些方面表现出相当大的差异。对于3-磷酸甘油酸的表观Km、其对抑制剂的敏感性以及其对盐和盐酸胍的反应尤其如此。然而，发现其Vmax与天然糖体PGK的Vmax相似。这些结果表明，C末端的变化导致了构象变化，影响了位于蛋白质N末端结构域的3-磷酸甘油酸结合位点。
