Pace C N, Barrett A J
Biochem J. 1984 Apr 15;219(2):411-7. doi: 10.1042/bj2190411.
We have used ribonuclease T1 and its chemically modified derivatives as substrates, and trypsin as proteinase, to investigate the kinetics of proteolysis of a specific peptide bond in the folded and unfolded conformations of a protein. Steady-state kinetic studies showed that Km = 0.27 mM and Kcat. = 2.45 s-1 for the tryptic hydrolysis of the Arg(77)-Val(78) peptide bond in unfolded ribonuclease T1. This Km is somewhat lower than, and the kcat. value similar to, values found for the tryptic hydrolysis of comparable bonds in small peptides. Our data for the initial velocity of hydrolysis of the Arg(77)-Val(78) bond in a solution of the folded protein indicate that the bond is at least 1700 times less rapidly hydrolysed in the folded than in the unfolded conformation of ribonuclease T1, and do not exclude the possibility that the bond is completely resistant to hydrolysis in the folded protein.
我们使用核糖核酸酶T1及其化学修饰衍生物作为底物，胰蛋白酶作为蛋白酶，来研究蛋白质折叠和未折叠构象中特定肽键的蛋白水解动力学。稳态动力学研究表明，对于未折叠的核糖核酸酶T1中Arg(77)-Val(78)肽键的胰蛋白酶水解，Km = 0.27 mM，Kcat. = 2.45 s-1。这个Km略低于小肽中可比键的胰蛋白酶水解的Km值，而kcat.值与之相似。我们关于折叠蛋白溶液中Arg(77)-Val(78)键水解初始速度的数据表明，该键在核糖核酸酶T1的折叠构象中的水解速度比未折叠构象中至少慢1700倍，并且不排除该键在折叠蛋白中完全抗水解的可能性。
