Structural and functional determinants of Mucor miehei protease VI. Inactivation of the enzyme by diazoacetyl norleucine methyl esters, pepstatin and 1,2-epoxy-30(p-nitro-phenyoxy)propane.
Mucor miehei protease (EC 3.4.21 -- ), an acid protease of fungal origin, was rapidly inhibited at pH 5.0 and 10 degrees C by a 78-fold molar excess of diazoacetyl norleucine methyl ester (N2Ac-Nle-OMe) when simultaneously added with a 78-fold molar excess of Cu(II). Preincubation with Cu(II) before the addition of N2Ac-Nle-OMe reduced the initial rate of activity loss presumably due to a copper-induced structural change as deduced from an examination of CD spectra. Cof norleucine and 1.02 +/- 0.041 mol of copper. The conformation of the N2Ac-Nle-OMe-inhibited enzyme appeared to be somewhat altered since the rate of H-3H exchange determined for the slowest exchanging class of hydrogens was reduced by more than 10-fold although the estimated number of hydrogens in this class remained constant. Mucor miehei protease was also inhibited by pepstatin; complete inactivation required a 6-fold molar excess of inhibitor and was associated with a major conformational change as determined from CD spectra. Loss of activity also occurred in the presence of 1,2-epoxy-3-(p-nitrophenyoxy)propane (EPNP).
D000614:Aminocaproates / Q000031:analogs & derivatives*; D001391:Azo Compounds / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology; D007700:Kinetics; D009089:Mucor / Q000201:enzymology*; D009596:Nitrophenols / Q000494:pharmacology*; D009646:Norleucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology*; D009994:Osmolar Concentration; D010436:Pepstatins / Q000494:pharmacology*; D011480:Protease Inhibitors; D013056:Spectrophotometry, Ultraviolet
D000614:Aminocaproates; D001391:Azo Compounds; D004852:Epoxy Compounds; D009596:Nitrophenols; D009842:Oligopeptides; D010436:Pepstatins; D011480:Protease Inhibitors; D003300:Copper; D009646:Norleucine; C007606:1,2-epoxy-3-(p-nitrophenoxy)propane