Juhász Tünde, Szeltner Zoltán, Polgár László
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest 112, Hungary.
Proteins. 2007 Nov 15;69(3):633-43. doi: 10.1002/prot.21522.
The peptidase domain of prolyl oligopeptidase is covered by a propeller domain, which excludes large peptides and proteins from the catalytic triad. Previous studies indicated that some amino acids of the N-terminal region constitute a part of the substrate entrance to the active site. To investigate the catalytic role of the N-terminus, we removed the residues 1-32 from the enzyme and examined the kinetic, thermodynamic, and structural consequences of the deletion, using the thermophile Pyrococcus furiosus prolyl oligopeptidase. An about threefold decrease in the catalytic activity along with a 20 degrees C reduction in the temperature optimum was observed. The pH-rate profile, the rate-limiting step, and the activation parameters did not change significantly. However, a substantial decrease was observed in the stability of the protein as demonstrated by circular dichroism and differential scanning calorimetry measurements, and by denaturation with guanidinium chloride. It was concluded that the N-terminal segment did not facilitate the substrate binding, independent of the size of the substrate, but contributed principally to the protein stability required for the formation of the proper active site.
脯氨酰寡肽酶的肽酶结构域被一个螺旋桨结构域覆盖，该结构域将大的肽和蛋白质排除在催化三联体外。先前的研究表明，N端区域的一些氨基酸构成了活性位点底物入口的一部分。为了研究N端的催化作用，我们从嗜热栖热菌脯氨酰寡肽酶中去除了1-32位残基，并研究了缺失这些残基对动力学、热力学和结构的影响。结果观察到催化活性下降了约三倍，最适温度降低了20℃。pH-速率曲线、限速步骤和活化参数没有显著变化。然而，通过圆二色性和差示扫描量热法测量以及用氯化胍变性表明，蛋白质的稳定性显著下降。得出的结论是，N端片段不促进底物结合，与底物大小无关，但其主要作用是为形成合适的活性位点所需的蛋白质稳定性做出贡献。
