Structural and functional determinants of Mucor miehei protease VI. Inactivation of the enzyme by diazoacetyl norleucine methyl ester, pepstatin and [formula omitted

Abstract

Mucor miehei protease (EC 3.4.23—), an acid protease of fungal origin, was rapidly inhibited at pH 5.0 and 10°C by a 78-fold molar excess of diazoacetyl norleucine methyl ester (N 2Ac-Nle-OMe) when simultaneously added with a 78-fold molar excess of Cu(II). Preincubation with Cu(II) before the addition of N 2Ac-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. Complete inactivation was associated with the incorporation of 1.6 ± 0.12 residues of norleucine and 1.02 ± 0.041 mol of copper. The conformation of the N 2Ac-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- nitrophenoxy)propane (EPNP).