The structure and function of acid proteases. IV. Inactivation of the acid protease from Mucor pusillus by acid protease-specific inhibitors.
Mucor pusillus acid protease was rapidly inactivated with 1 : 1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was maximal at around pH 6 when the enzyme was mixed with DAN and cupric ions without prior mixing of the reagents, and at pH 5.3 when DAN and cupric ions were mixed and incubated before addition to the enzyme solution. In both cases, the rate of inactivation decreased as the pH was either increased or decreased. The amino acid composition of an acid hydrolysate of the DAN-Modified enzyme was indistinguishable from that of the native enzyme except for the incorporation of about one norleucine residue per molecule of protein. The enzyme was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). At the stage of about 90% inactivation, 1.50 residues of EPNP were incorporated per molecule of protein and the rate of inactivation followed pseudo-first order kinetics. The optimal pH for the inactivation was pH 3.0 and the rate of inactivation decreased as the pH was either increased or decreased. Furthermore, the enzyme was strongly inhibited by pepstatin, and the reactions of DAN and of EPNP was also inhibited significantly by prior treatment of the enzyme with pepstatin. These results suggest that the enzyme may have two essential carboxyl groups at the active site, one reactive with DAN in the presence of cupric ions and the other with EPNP, and that pepstatin binds part of the active site to inhibit the reactions with DAN and EPNP as well as the enzyme activity.
D000614:Aminocaproates / Q000494:pharmacology*; D001391:Azo Compounds / Q000494:pharmacology; D001665:Binding Sites; D003300:Copper / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009089:Mucor / Q000201:enzymology*; D009596:Nitrophenols / Q000494:pharmacology; D009646:Norleucine / Q000031:analogs & derivatives / Q000494:pharmacology*; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors
D000614:Aminocaproates; D001391:Azo Compounds; D009596:Nitrophenols; D010436:Pepstatins; D011480:Protease Inhibitors; D003300:Copper; D009646:Norleucine