The structure and function of acid proteases. VI. Effects of acid protease-specific inhibitors on the acid proteases from Aspergillus niger var. macrosporus.
1. The Type B acid protease from Aspergillus niger var. macrosporus was inactivated by reaction with diazoacetyl-DL-norleucine methyl ester (DAN), DL-1-diazo-3-tosylamido-2-heptanone (DTH), and L-1-diazo-3-tosylamido-4-phenyl-2-butanone (DTPB) in the presence of cupric ions. The reaction with DAN took place with 1:1 stoichiometry. The enzyme was also inactivated by reaction with 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) with concomitant incorporation of approximately two EPNP molecules per molecule of protein. Moreover, these reactions of DAN and of EPNP were markedly inhibited by pepstatin. These results seem to indicate that, as in the case of porcine pepsin [EC 3.4.23.1] and related acid proteases, the enzyme has two essential carboxyl groups at the active site, one reactive with DAN and related diazo reagents in the presence of cupric ions and the other reactive with EPNP, and that pepstatin binds in the vicinity of these residues. 2. The Type A acid protease from the same mold, on the other hand, was found to be markedly less sensitive to these specific inhibitors. Under conditions where the Type B enzyme was completely inactivated by DAN and related diazo reagents, only partial inactivation of this enzyme occurred. The effect of prior mixing of DAN and cupric ions on the pH profile of inactivation was also different from that for the Type B enzyme. Moreover, the Type A enzyme was not inactivated by EPNP. These results thus indicate that the nature of the active site of the Type A enzyme is rather different from that of the Type B enzyme and hence that the Type A enzyme belongs to a different class of acid proteases from the Type B enzyme.
D001230:Aspergillus / Q000201:enzymology*; D001234:Aspergillus niger / Q000201:enzymology*; D001391:Azo Compounds / Q000494:pharmacology; D001665:Binding Sites; D003300:Copper / Q000494:pharmacology; D003979:Diazonium Compounds / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009596:Nitrophenols / Q000494:pharmacology; D009646:Norleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors
D001391:Azo Compounds; D003979:Diazonium Compounds; D004852:Epoxy Compounds; D009596:Nitrophenols; D010436:Pepstatins; D011480:Protease Inhibitors; D003300:Copper; D009646:Norleucine