Structural study on the active site of porcine pepsin and Rhizopus chinensis acid protease. Spin labeling with diazoketone reagents.
To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2.
D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010434:Pepsin A / Q000378:metabolism*; D010436:Pepstatins / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012233:Rhizopus / Q000201:enzymology*; D013552:Swine
D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; C031375:pepstatin