Kinetics of tryptic hydrolysis of the arginine-valine bond in folded and unfolded ribonuclease T1.
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.
D001120:Arginine / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D010455:Peptides; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism*; D014633:Valine / Q000378:metabolism
D010455:Peptides; D001120:Arginine; D004722:Endoribonucleases; D006163:Ribonuclease T1; D014357:Trypsin; D014633:Valine